A novel mechanism, linked to cell density, largely ... · 1 1 A novel mechanism, linked to cell...

1

A novel mechanism linked to cell density largely controls cell 1

division in Synechocystis 2

3 Alberto A Esteves-Ferreira12 Masami Inaba1 Toshihiro Obata3 Antoine Fort4 Gerard 4

Fleming5 Wagner L Arauacutejo6 Alisdair R Fernie3 and Ronan Sulpice1 5

6

1 National University of Ireland - Galway Plant Systems Biology Lab Plant and 7

AgriBiosciences Research Centre School of Natural Sciences Galway Ireland 8

2 CAPES Foundation Ministry of Education of Brazil Brasilia DF 70040 020 Brazil 9

3Central Metabolism Lab Department of Molecular Physiology Max Planck Institute of 10

Molecular Plant Physiology Max Planck Society 11

4National University of Ireland - Galway Genetics amp Biotechnology Research lab Plant 12

and AgriBiosciences Research Centre School of Natural Sciences Galway Ireland 13

5National University of Ireland ndash Galway Microbiology School of Natural Sciences 14

Galway Ireland 15

6Max-Planck Partner group at the Departamento de Biologia Vegetal Universidade 16

Federal de Viccedilosa 36570-900 Viccedilosa Minas Gerais Brasil 17

18 One sentence summary Nutrient limitation self shading and quorum sensing are not 19 major limiting factors for the growth of Synechocysits in batch cultures 20 21 Running title Factors limiting the growth of Synechocystis 22 23 List of Author contributions 24 AAE-F and RS conceived the original research plans AAE-F and TO performed 25 most of the experiments MI AF and GF provided technical assistance and helped 26 for design of experiments AAE-F and RS designed the experiments and analysed the 27 data AAE-F and RS conceived the project and wrote the article with contributions of 28 all the authors ARF GF and WLA supervised and complemented the writing 29 30

Plant Physiology Preview Published on June 26 2017 as DOI101104pp1700729

Copyright 2017 by the American Society of Plant Biologists

wwwplantphysiolorgon November 16 2018 - Published by Downloaded from Copyright copy 2017 American Society of Plant Biologists All rights reserved

2

Funding information 31 AAE-F is recipient of Scholarships granted by the Agency for the Support and 32

Evaluation of Graduate Education (CAPES-Brazil ndash grant BEX 890413-8) and the 33

Thomas Crawford Hayes (NUI Galway) RS is recipient of a Visiting Professor grant 34

from (CNPq-Brazil PVPE grant 4010902014-0 - Brazil) and the Millenium fund 35

(NUI Galway) 36

37 Corresponding author Dr Ronan Sulpice Tel +353-(0)-91494292 Email 38

ronansulpicenuigalwayie 39

40 41 42

43


1

ABSTRACT 44

Many studies have investigated the various genetic and environmental factors 45

regulating cyanobacterial growth Here we investigated the growth and metabolism of 46

Synechocystis sp PCC 6803 under different nitrogen sources light intensities and CO2 47

concentrations Cells grown on urea showed the highest growth rates However for all 48

the conditions the daily growth rates in batch cultures decreased steadily over time and 49

stationary phase was obtained with similar cell densities Unexpectedly metabolic and 50

physiological analyses showed that growth rates during log phase were not primarily 51

controlled by the availability of photoassimilates Further physiological investigations 52

indicated that nutrient limitation quorum sensing light quality and intensity (self-53

shading) were not the main factors responsible for the decrease in the growth rate and 54

for the onset of the stationary phase Moreover cell division rates in fed-batch cultures 55

were positively correlated with the dilution rates Hence not only light CO2 and 56

nutrients can affect growth but also a cell-to-cell interaction Accordingly we propose 57

that cell-cell interaction may be a factor responsible for the gradual decrease of growth 58

rates in batch cultures during log phase culminating with the onset of stationary phase59


1

INTRODUCTION 60

Cyanobacteria were the first organisms on Earth that performed oxygenic 61

photosynthesis and nowadays they are widespread on a variety of terrestrial and aquatic 62

habitats showing extensive metabolic physiological and morphological diversity 63

(Tomitani et al 2006 Beck et al 2012 Schirrmeister et al 2013) This diversity 64

among cyanobacteria has been related with their capability to adapt to series of changes 65

which occurred on earth affecting the availability of the atmospheric oxygen light 66

carbon dioxide (CO2) and reduced nitrogen (Catling et al 2001 Herrero et al 2001 67

Kasting and Siefert 2002 Kuhl et al 2005 Muramatsu and Hihara 2011 Hagemann 68

et al 2016) 69

Synechocystis sp PCC 6803 (hereafter termed Synechocystis) is one of the 70

cyanobacterial species commonly used for metabolic and genetic studies It is easy to 71

culture under phototrophic mixotrophic and heterotrophic conditions its entire genome 72

is sequenced and exhibits natural competence for incorporation of exogenous DNA 73

(Kanesaki et al 2012 Yu et al 2013) Like all photosynthetic organisms the 74

physiology and cell cycle of Synechocystis is affected both by light availability and 75

intensity (Yang et al 2010 Schuurmans et al 2015) Light harvesting is performed by 76

phycobilisomes composed of a core of allophycocyanins and branches of phycocyanin 77

(Mullineaux 2008 Arteni et al 2009) The light energy is transferred from the 78

phycobilisomes to chlorophyll a present in the reaction centers of photosystems I (PSI) 79

and II (PSII) (Liu et al 2013) These transfers lead to the generation of reducing power 80

which supplies metabolic processes such as the Calvin-Benson cycle and nitrogen 81

uptake and reduction (Valladares et al 2002 Flores et al 2005) The efficiency of CO2 82

fixation and assimilation is at least partially under the control of a carbon 83

concentration mechanism (CCM) which increases the CO2 concentration around the 84


2

Rubisco catalytic site (Price et al 1998) allowing it to operate close to its Vmax (Badger 85

et al 2006) Synechocystis can uptake and metabolize different nitrogen sources 86

(nitrate nitrite ammonium urea and some amino acids Quintero et al 2000 87

Valladares et al 2002 Flores et al 2005 Muro-Pastor et al 2005) Intriguingly the 88

control of the expression and activity of transporters and enzymes related with nitrogen 89

metabolism is regulated by a complex network involving 2-oxoglutarate NtcA 90

(nitrogen control factor of cyanobacteria) PII (nitrogen regulatory protein PII) and PipX 91

(PII interacting protein X) (Alfonso et al 2001 Herrero et al 2001 Llaacutecer et al 2010 92

Zhao et al 2010 Espinosa et al 2014 Luumlddecke and Forchhammer 2015) 93

In photo-bioreactors Synechocystis can typically achieve growth rates ranging 94

from 17 to 25 divisions day-1 ie doubling times of between 141 and 96 hours (Kim 95

et al 2011) Faster doubling times of 6 and 56 hours were reported by Hihara et al 96

(2001) and Zavřel et al (2015) respectively however in the case of the latter study 97

these division rates could not be maintained for more 120 hours Many studies have 98

attempted to identify growth limiting factors in Synechocystis (eg nutrients and light 99

Kim et al 2011 Lea-Smith et al 2014 Burnap 2015 Touloupakis et al 2015 van 100

Alphen and Hellingwerf 2015) Understanding of the factors controlling the limitation 101

of Synechocystis growth would facilitate use this strain as a cell factory (Yu et al 2013) 102

for the production of biomass (Joseph et al 2014) pigments (Sekar and 103

Chandramohan 2008) secondary metabolite natural products (Frommeyer et al 2016) 104

biofuel (Dexter and Fu 2009 Baebprasert et al 2011 Liu et al 2011) and other high 105

value compounds 106

The commercialisation of cyanobacteria-based biomass and biomolecules still 107

requires optimization for sustainable economic viability (Schenk et al 2008 Brennan 108

and Owende 2010 Wijffels et al 2013) One recently adopted approach in this view is 109


3

the use of low cost sources of CO2 nutrients and water (Markou and Georgakakis 110

2011 Slade and Bauen 2013 Iijima et al 2015) In this study we grew Synechocystis 111

in various growth conditions using three different nitrogen sources in combination 112

with two light intensities and two CO2 concentrations Urea is seemingly a potent 113

source of nitrogen supporting growth of Synechocystis as it promoted high cell division 114

rates and is less costly than nitrate and ammonium That said after four days of culture 115

growth was inhibited for all conditions tested and this inhibition was not related to a 116

metabolic limitation Indeed we observed an uncoupling of photosynthesis and growth 117

leading to the accumulation of unused reserves in the cells and even their release to 118

culture media We further investigated the reasons behind this non-metabolic growth 119

limitation and conclude that nutrient limitation and quorum sensing are not responsible 120

for the decrease of growth rates during the log phase and for the onset of the stationary 121

phase Our data also suggest that self-shading is not responsible for the growth 122

limitation We hypothesise that Synechocystis may be able to sense the gradual increase 123

of cell density occurring in batch cultures via cell-cell interaction leading to gradual 124

decrease of division rate until the onset of stationary phase 125

126


4

MATERIAL AND METHODS 127

We selected the strain Synechocystis sp PCC 6803 for our study since it is a 128

major cyanobacterial model with very large amount of literature available about its 129

physiology and molecular characteristics The strain was obtained directly from the 130

French Pasteur Culture Collection (PCC strain) and shows positive phototaxis when 131

grown on BG-11 agar plates (Kanesaki et al 2012) 132

Growth conditions 133

Synechocystis was cultivated under two light intensities (50 and 200 micromol 134

photons m-2 s-1) two CO2 concentrations (ambient and 25 mM bicarbonate) and three 135

nitrogen sources (nitrate ammonium and urea) for a total of ten different growth 136

conditions All cultures were grown under 18h6h daynight photoperiod at a constant 137

temperature of 26deg C plusmn 1deg C and constantly shaken at 120 rpm To allow complete 138

acclimation to each treatment Synechocystis was cultivated 3 times from log to 139

stationary phase in all growth conditions before analyses were performed 140

Cultures were initiated with 3 mL of inoculum in a conical flask (500 mL) 141

containing 147 mL of BG-11 medium (2 vv) (Rippka et al 1979) for a starting cell 142

density ranging from 4 to 6 x 106 cells mL-1 BG-110 (without nitrogen) was 143

supplemented with sodium nitrate (177 mM) or ammonium (5 mM) or urea (25 mM) 144

Due to the release of CO2 when urea is hydrolysed the same concentration of CO2 145

(25mM) was added in ammonium and nitrate cultures in the form of sodium 146

bicarbonate (NaHCO3 ndash hereafter CO2) To maintain the pH at 75 plusmn 05 HEPES (20 147

mM) was added in the media 148

Estimation of generation time 149

Growth cycle was evaluated by counting cells in a Neubauer chamber (Optik 150

Labor Germany) at intervals of 24 h till the third consequent day showing no 151


5

significant increases in cell density (stationary phase) The generation time (Gt) for each 152

treatment was calculated by using a regression of the data within the logarithmic growth 153

period 154

Experimental set-ups to test nutrient limitation quorum sensing and self-shading 155

To evaluate the possibility that nutrient depletion andor quorum sensing affect 156

cell density at stationary phase Synechocystis was cultured for 15 days in the different 157

growth conditions described above in order to reach stationary phase Then all the 158

volume was centrifuged The pellet was resuspended in a fresh BG-11 media (new BG-159

11) and the volume adjusted to 150 mL The supernatant (old BG-11) was filtered on a 160

nitrocellulose membrane (47 mm x 022 microm Sarstedt) and inoculated with 3 mL of the 161

respective resuspended pellet Cell density was determined and cultures were grown in 162

the same conditions as previously After four days of cultivation the cellular density was 163

determined to assess growth rates 164

One growth condition urea as the nitrogen source and the light intensity of 200 165

micromol photons m-2 s-1 for which maximum growth rates were achieved was selected to 166

evaluate how many growth cycles of Synechocystis BG-11 could support Synechocystis 167

was cultured during 8 days in fresh BG-11 till onset of stationary phase Subsequently 168

the entire culture was centrifuged and the supernatant (old BG-11) was filtered and used 169

to grow an inoculum obtained from the resuspended pellet for additional 8 days At the 170

end of this period cultures were centrifuged and the supernatant was filtered and reused 171

again (reused old BG-11) to culture for 8 days an inoculum obtained from previous 172

pellet Finally cultures were divided in two equal volumes and supplemented in a 173

proportion of 11 with fresh BG-11 or water and kept growing for 8 more days 174

To test for quorum sensing and light qualityintensity cultures of Synechocystis 175

were cultivated on urea at 50 or 200 micromol photons m2 s-1 for 15 days in order to obtain a 176


6

culture at saturation phase For both light intensities 3 translucent microtubes (2 mL) 177

were filled with 15 mL of BG-11 culture media (supplemented with urea) containing 178

2 (vv) of the respective Synechocystis culture These tubes were glued at the bottom 179

of conical flasks (500 mL) after being sealed with filters (Sigma Iso-Disctrade Filters 180

PFTE-4-4 4 mm x 045 microm) The filters allowed for media exchanges but cells could 181

not go in or out Then the conical flasks were filled with 150 mL of the respective 182

Synechocystis culture at stationary phase or with fresh BG-11 culture media The flasks 183

were kept shaking for 4 days in the same light intensity the cultures grew before After 184

four days of culture the cellular density in the tubes was determined 185

Experimental set-ups to evaluate the effect of cell density on growth 186

To investigate whether a cell-cell interaction can affect negatively growth rate of 187

Synechocystis and how the effect is related quantitatively to the cell density of the 188

culture three different experiments were performed All the experiments were 189

performed with cultures on urea under 200 micromol photons m-2 s-1 where maximum 190

growth rates were achieved 191

In the first experiment growth cycle of Synechocystis was evaluated in cultures 192

starting with four different initial cell densities 2 x 106 4 x 106 6 x 106 and 2 x 107 193

cells mL-1 The growth cycle and Gt of each treatment was calculated as described 194

before 195

In the second experiment cell density was artificially increased by 10 times 196

during log phase For that cultures of 150 mL were grown until a density of 1 x 107 cells 197

mL-1 was reached They were then separated in three groups (1) cultures were used as 198

controls (ie they were maintained growing) (2) cultures were centrifuged and 199

concentrated 10 times to a final density of 1 x 108 cells mL-1 and (3) cultures were 200

submitted to the same pipetting and centrifuging steps than in group 2 but resuspended 201


7

without being concentrated (centrifuged controls) The growth of the three groups was 202

then monitored daily as described above 203

In the third experiment Synechocystis was grown in fed-batch conditions to 204

evaluate how its growth responds to different dilution rates and cell densities Firstly 205

cultures were grown as batch to assess if their growth properties in a photoreactor were 206

similar to the ones observed previously in conical flasks Once cultures reached 207

stationary phase with a similar growth cycle as previously cultures were diluted at a 208

density of 4 x 106 cells mL-1 After 24 hours the pump started to drop 56 mL of media 209

per hour working only during the 18 hours of the light period which meant a daily 210

volume of 100 mL of media and a daily dilution rate of 05 time as the initial volume of 211

culture in the photoreactor was 200 ml Every day at the beginning of the light period 212

the volume of the cultures was readjusted to the initial volume of 200 mL A second 213

daily dilution rate of 2 times was also tested by dropping 334 mL of media per hour 214

during the light period Both dilution experiments were also performed with cultures at 215

stationary phase (2 x 108 cells mL-1) 216

Biomass photosynthesis metabolic profile and gene expression determination 217

All the physiological metabolic and molecular analyses were performed with 218

samples harvested at day four (log phase) and three days after the onset of the stationary 219

phase both at the end of the day and night 220

Dry Biomass 221

Biomass was determined by filtering 50 mL of cultures on nitrocellulose 222

membranes which had been previously weighted The material was dried at 70 ordmC for 223

48 hours and then maintained for 24 hours in a desiccation chamber with blue silica 224

gel The weight of the membrane plus biomass was then determined and the dry 225

biomass weight was obtained by subtraction of the membrane weight 226


8

Oxygen evolution analysis 227

Photosynthesis was analyzed using non-invasive optical oxygen sensors 228

(PreSens - Germany) Oxygen sensor spots were glued inside the conical flasks (500 229

mL) so that they were in contact with the culture The instrument was calibrated using a 230

solution of sodium hydrosulphite to set 0 saturation and after with distilled water 231

Analysis was started at the end of the dark period and after obtaining a stable readout of 232

oxygen levels the light was switched on Oxygen evolution was then measured for at 233

least 10 minutes 234

Determination of metabolite contents 235

Samples were collected with a syringe filled with a quenching solution (60 vv 236

methanol in water) precooled to lt-20degC The solutions were then quickly transferred to 237

centrifuge tubes stored in an ice bath Each quenched sample was centrifuged for 20 238

minutes at 4000 rpm (Young et al 2011) Pellets were subjected to hot ethanol 239

extraction (Cross et al 2006) The soluble fraction was used for the determination of 240

chlorophyll a (Ritchie 2006) and total amino acid contents (Schippers et al 2008) In 241

the insoluble fraction protein and glycogen contents were determined according to 242

Lowry et al (1951) and Hendriks et al (2003) respectively Total soluble sugars were 243

determined as described previously (Dubois et al 1956) The determination of a large 244

set of primary metabolites was performed by gas chromatography coupled with mass 245

spectrometry (GC-MS) (Krall et al 2009) using palatinose as an internal standard For 246

cyanophycin and phycocyanin determination samples were sonicated (40 amplitude 247

for 4 minutes) and subsequently centrifuged for 40 minutes at 4000 rpm Phycocyanin 248

was determined from the supernatant (Tandeau de Marsac 1977) and cyanophycin was 249

extracted from the remaining pellet as previous described (Burnat et al 2014) and 250

determined by Sakaguchi reaction (Messineo 1966) 251


9

Gene expression analysis 252

RNA was extracted according to Kim et al (2006) applying the freeze-thaw 253

step described by Kim et al (2012) before disrupting the cells in a Tissuelyser (Qiagen) 254

RNA concentration was quantified spectrophotometrically at 260 nm and its integrity 255

was checked by electrophoresis on a denaturing agarose gel (16 mv) (Goda and 256

Minton 1995) Due to the presence of DNA samples were treated with RNase-free 257

DNase I (Sigma) according to the manufacturerrsquos instructions For cDNA synthesis 258

1000 ng of total RNA was reverse transcribed with SensiFAST cDNA Synthesis Kit 259

(Bioline) in a final volume of 20 microl following the manufacturerrsquos instructions 260

Subsequently cDNA was diluted 3 times and stored at -20deg C The RT-qPCRs were 261

performed on 96-well PCR plates covered with a sealing tape (Thermo) using 262

SensiMixTM SYBRreg No-ROX Kit (Bioline) following the manufacturerrsquos instructions 263

To calculate the relative expression levels of ntcA pII and ftsZ (cell division protein 264

FtsZ) rnpB (RNA subunit of ribonuclease P) was selected as the reference gene as 265

previously described (Pinto et al 2012) 266

Experimental design and statistical analysis 267

The experiments were performed and analyzed according to a randomized block 268

design (RBD) with three replications The data obtained at the end of the day and night 269

were subjected to t-test at 5 probability Analysis of variance (ANOVA P lt 005) 270

were performed to compare the data obtained for the different growth conditions and 271

the means were compared by Tukey test at 5 probability using the lowest value as a 272

reference All these analyses were performed with the software package SPSS 22 273

Windows amp Macintosh (ArmonkNew York) Finally the results obtained were 274

independently analyzed via Spearmanrsquos correlation using Excel 2013 (Microsoft) 275

276


10

277


11

RESULTS 278

Rates of cell division peak at start of log phase and then gradually decrease 279

The growth cycle and Gt of Synechocystis varied in response to the nitrogen 280

source light intensity and CO2 concentration (Fig 1 Table I) A lag phase was not 281

observed when cells were grown in the presence of ammonium under moderate light 282

intensity (ML 50 micromol photons m-2 s-1) nitrate under ML with CO2 supplementation 283

and nitrate under high light intensity (HL 200 micromol photons m-2 s-1 Fig 1A-D) The 284

highest number of divisions per day were typically achieved at day two or three and 285

gradually decreased afterwards (Fig 1B D and F) The cultures reached a stable 286

stationary phase after 8 to 14 days (Fig 1) Curiously despite a large variation in the 287

duration of the growth cycles in nine out of the ten growth conditions studied the 288

stationary phase occurred at similar cell densities (21 to 26 x 108 Fig 1 Supplemental 289

Table S1) In order to confirm whether cells were in stationary phase we analysed the 290

expression of ftsZ gene which encodes the cell division protein FtsZ (Supplemental Fig 291

S1) The relative ftsZ expression levels of the cells at stationary phase at the end of the 292

day (ED) were 4 to 10 times lower than the levels observed for cells in log phase 293

(Supplemental Fig S1(A)) In addition as observed previously (Mori and Johnson 294

2001) the ftsZ expression levels were very low at the end of the night (EN) with little 295

differences between the growth stages (Supplemental Fig S1(B)) thus confirming that 296

cells do not divide at night 297

The gradual decrease in the number of division per day observed from the third 298

or fourth day of culture and the fact that cultures reached stationary phase with 299

comparable cell densities regardless of differences in nitrogen source light intensity and 300

CO2 concentration raised questions about a similar metabolic limitation occurring for all 301

these growth conditions due to (1) nutrient starvation (2) a reduction in light intensity 302


12

due to high cell density (self-shading) (3) or a regulatory limitation of growth due to 303

quorum sensing or light quality In order to test these hypotheses we analysed the 304

photosynthetic rates cellular metabolic contents and evaluated the possibilities of 305

nutrient depletion quorum sensing and self-shading 306

Metabolic and physiological traits during the log phase 307

The varied growth conditions did not affect the Synechocystis cell size 308

(Supplemental Table S2) Then as our aim was to document the metabolic contents of 309

the cells and relate this information to growth rates we normalized all our data on a per 310

cell basis and not on a biomass basis in order to avoid biases due to specific growth 311

conditions leading to cells containing more metabolites and then being heavier Indeed 312

expressing our data on a biomass basis would have wrongly led to the conclusion of 313

higher growth rates for some growth conditions whilst in fact it could just be caused by 314

cells accumulating more metabolites 315

The rate of photosynthesis as estimated by the determination of oxygen release 316

varied between the treatments (Table II) Under HL cells grown in the presence of 317

nitrate supplemented with CO2 ammonium and urea exhibited the highest 318

photosynthetic rates (Table II) In addition photosynthetic rates of cells cultured with 319

ammonium and urea showed a light effect with an increase by almost 60 when grown 320

under HL (Table II) By contrast nitrate cultures displayed similar photosynthetic rates 321

and were not affected by increases in light intensity or CO2 supplementation (Table II) 322

After four days of culture the cellular dry weight (hereafter termed biomass) was 323

higher under ML than HL for all the treatments except for ammonium without CO2 324

supplementation for which the biomass did not differ (Supplemental Table S3) 325

Chlorophyll a (hereafter termed chlorophyll) content was affected by nitrogen source 326

and mostly by light intensity In general its content was higher under ML in particular 327


13

for cultures supplemented with nitrate (Supplemental Fig S2(A)) The highest 328

concentrations of phycocyanin were registered for cultures grown in the presence of 329

nitrate and urea under ML and the lowest for cultures grown in the presence of 330

ammonium and nitrate under HL (Supplemental Fig S2(B)) Levels of total soluble 331

sugars were almost constant among the different growth conditions (Supplemental Fig 332

S3(A)) As previously documented (Mikkat et al 1997) sucrose levels were below the 333

level of detection with the exception of cells grown on nitrate and the presence or 334

absence of CO2 under ML (Supplemental Table S4 and S5) Total soluble sugars were 335

also detected in the growth media however their concentrations were similar for almost 336

all treatments (Supplemental Fig S4) Glycogen amounts at ED were relatively constant 337

for all growth conditions and only cells grown under HL and supplemented with CO2 338

accumulated significantly more glycogen independently of the nitrogen source 339

(ammonium or nitrate Supplemental Fig S3(B)) A significant amount of the glycogen 340

accumulated at ED was used during the night for all growth conditions (from 171 to 341

666) with the exception of cells grown in the presence of nitrate with ML 342

(Supplemental Fig S3(B) and S5) Levels of total soluble amino acids were very stable 343

in cells grown under ML and did not exhibit diurnal variation In contrast their levels 344

showed large variations between the treatments under HL (Supplemental Fig S3(C)) 345

The cellular protein content did not vary diurnally and was relatively similar between 346

ammonium and urea growth conditions Cells grown on nitrate displayed significantly 347

higher protein content (Supplemental Fig S3(D)) Cyanophycin levels did not vary 348

diurnally and showed small variations between all growth conditions (Supplemental 349

Fig S3(E)) 350

In order to obtain insights into the carbon-nitrogen (CN) balance and cellular 351

redox status of the cells grown in the ten contrasting growth conditions we determined 352


14

ntcA and pII expression (Supplemental Fig S6) The relative expression levels of both 353

genes showed no significant variations in response to the nitrogen source and CO2 354

concentrations On the other hand their levels of expression at ED were negatively 355

affected by the increase of the light intensity (Supplemental Fig S6) In addition ntcA 356

and pII exhibit a significant reduction in their expressions at EN for all treatments 357

(Supplemental Fig S6) 358

Metabolic and physiological traits during the stationary phase 359

For most growth conditions photosynthetic rates decreased by 50-60 three 360

days after the onset of the stationary phase (Table II) thus allowing the maintenance of 361

a significant production of photoassimilates Moreover cells cultured in the presence of 362

nitrate and ammonium (not supplemented with CO2) under HL kept similar rates of 363

photosynthesis with those observed in log phase (Table II) 364

Biomass of cultures under ML exhibited a lower variation than at HL 365

(Supplemental Table S3) The levels of chlorophyll and phycocyanin were similar in 366

almost all of the growth conditions (Supplemental Fig S7) Higher contents of total 367

soluble sugars were observed for all cells grown under HL especially for those grown 368

in presence of nitrate plus CO2 and ammonium (Supplemental Fig S8(A)) This makes 369

sense as these growth conditions were the ones in which photosynthetic rates remained 370

high at stationary phase (Table II) By contrast higher sucrose levels were determined 371

under ML (Supplemental Fig 8(B) Tables S6 and S7) The highest levels of glycogen 372

were observed in cultures grown in the presence of nitrate under ML Glycogen 373

degradation was significant for most of the growth conditions under HL (Supplemental 374

Fig S8(C)) As the photosynthetic rates were two times higher for some growth 375

conditions without proportional increase of total soluble sugars and glycogen cellular 376

contents (Supplemental Fig S8(A) (C)) we next analysed total soluble sugars in the 377


15

culture media The highest levels of sugars in media were measured for the three growth 378

conditions where photosynthetic rates were maintained (Supplemental Fig S9) 379

suggesting that photosynthetic rates were higher than necessary for the growth demand 380

and that the excess of photosynthate production is released to the media Total soluble 381

amino acids content was also increased in cells grown under HL independently of the 382

nutrient treatments (Supplemental Fig S10(A)) In good agreement with this glutamate 383

levels were lower in these growth conditions (Supplemental Fig S10(B) Tables S6 and 384

S7) In addition light intensity negatively affected cyanophycin accumulation 385

(Supplemental Fig S10(C)) whilst all treatments displayed similar protein contents 386

(Supplemental Fig S10(D)) 387

The decrease in division rates seem to be unrelated to nutrient limitation quorum 388

sensing or self-shading 389

To evaluate the possible influence of nutrient depletion andor quorum sensing 390

we transferred cells from a whole culture which had reached stationary phase to fresh 391

media (Fig 2) Cell density did not increase significantly (Table III Supplemental 392

Tables S8 and S9) In order to assess if the old media obtained from the culture which 393

had reached stationary phase was sub-optimal for growth we grew a 3 mL inoculum 394

from the same resuspended pellet in the old media (Fig 2) After four days of culture 395

cell density increased 11 to 22 times with the division rates being statistically similar to 396

the rates observed when an inoculum of the same density was gown on fresh media 397

(Table III Supplemental Tables S8 and S9) Besides the whole growth cycle observed 398

using the old media appeared very similar to the one observed on fresh media and only 399

when we attempted to re-use the media for a third growth cycle we could observe a 400

sharp decrease in division rates High daily cell division rates were restored by addition 401

of fresh nutrients to the media but not by addition of water (Fig 3) Thus the slow 402


16

growth rates at stationary phase seems likely not due to a nutrient limitation as our 403

metabolic data suggested nor to quorum sensing as the growth of an inoculum would 404

have been impaired in old media due to the presence of a signal molecule though we 405

cannot exclude the possibility that this molecule had been highly unstable 406

To confirm the absence of quorum sensing in our culture conditions and test also 407

the possibility of an effect of light intensity andor quality due to the high density of the 408

cell culture we next inoculated cells into a microtube fixed at the bottom of a flask 409

containing a culture which had reached stationary phase (Fig 2) Importantly we 410

allowed exchange of media between the culture and the cells within the microtube by 411

sealing a filter of 045 microm at the entrance of the microtube After four days of culture 412

under ML cell density in the microtubes reached 60 of what was achieved by an 413

inoculum in presence of fresh media (Table IV) so the cells divided only one time less 414

Moreover when cells were grown under HL we did not observe any difference with a 415

fresh culture (Table IV) This indicates that quorum sensing is not responsible for the 416

decrease of growth rates and for the onset of stationary phase in Synechocystis 417

Moreover self-shading had a very moderate effect under ML and probably no effect 418

under HL (Fig 1 Supplemental Table S1) 419

Given that nutrient limitation quorum sensing and self-shading may not explain 420

the gradual decrease of growth and the onset of stationary phase we considered whether 421

a regulation triggered by cell-cell interaction could be involved in this process For this 422

purpose experiments were performed with cultures grown on urea under HL to avoid 423

the moderate self-shading effect observed under ML In order to test this hypothesis we 424

started cultures using inoculums at different cell densities Cultures initiated with higher 425

cell density showed slower division rates during the second and third days of culturing 426


17

and all cultures exhibited a gradual decrease in their division rates from the fourth day 427

(Fig 4A and B) 428

In order to evaluate how fast the rate of cell division responded to an increase in 429

cell density we used centrifugation to artificially increase by a factor of 10 the density 430

of log phase cultures This treatment led to a 58 and 77 decrease in growth rates 431

after 24 and 48 hours respectively Cultures submitted to the same centrifugation 432

process but resuspended without being concentrated did not present any alteration in 433

their growth rate dividing as fast as cultures which had not been centrifuged (Fig 4C 434

and D) 435

In order to determine whether cell density is quantitatively controlling the rate of 436

cell division we grew Synechocystis in a fed-batch system at two different dilution 437

rates Cultures started with low cell density (4 x 106 cells mL-1) and submitted to a 438

dilution rate of 05 time increased in density during the first four days (Fig 5A and B) 439

When cultures reached a density of 7 x 107 cells mL-1 growth and dilution rate were 440

counter-balanced and the culture density stayed stable with cells dividing around 046 441

to 066 time per day for seven days (Fig 5A and B) Next we increased the dilution rate 442

to 2 times per day The cells divided faster than the dilution rate during the first two 443

days till the culture reached a density of 35 x 107 cells mL-1 when cell division rate and 444

dilution rate were counter-balanced During the following days the cells divided fast 445

with 17 to 22 divisions per day (Fig 5A and B) 446

Next we investigated the effect of the same dilution rates on cultures which had 447

reached stationary phase At a dilution rate of 05 time per day cultures divided slower 448

than the dilution rate in the first day leading to a decrease of the cell density (Fig 5C 449

and D) Then cell division rates entered in balance with dilution dividing 046 to 074 450

division per day during the following 6 days When we applied a dilution rate of 2 times 451


18

per day we observed a gradual increase in cell division rates which counter-balanced 452

the dilution rate on the third day of growth at a cell density around 2 x 107 cells mL-1 453

with cells diving 16 to 23 times per day for the next 6 days (Fig 5C and D) In 454

summary cell division rates appeared to be directly related to the cell density of the 455

cultures 456

457


19

458


20

DISCUSSION 459

Growth rates are only partially controlled by the metabolic capacity and mostly by 460

the cell division machinery 461

We built a Spearmanrsquos rank correlation matrix to identify potential metabolic 462

and physiological traits which could be linked to growth and biomass (Fig 6 463

Supplemental Fig S11) Gt and biomass were significantly and positively correlated (rs 464

= 071 Fig 6) meaning that cells dividing fast do accumulate less biomass than cells 465

dividing slowly Photosynthesis did not correlate with growth and biomass However 466

photosynthetic rates were positively correlated with amino acids levels at both ED and 467

EN and with soluble sugars pools and glycogen at EN (Fig 6) The absence of 468

correlation between Gt and A (photosynthetic rate) indicates that photosynthesis and 469

growth were unrelated in the fourth day of culture so three days after the start of the log 470

phase for most of the culture conditions studied This suggests that instead of dividing 471

the cells store photoassimilates (Fig 6 and 7) In agreement Gt correlates positively 472

with proteins and sucrose at ED and EN and with glycogen soluble sugars citrate and 473

malate at EN Additionally we observed a strong negative correlation between cell 474

number with protein and total soluble sugars both at ED and EN (Fig 6) Thus the 475

gradual decrease in the division rate observed in our batch cultures (Fig 1) does not 476

appear to be due to a metabolic limitation but to a progressive decrease in the ability of 477

cells to divide Altogether we observe uncoupling of Gt and A a progressive increase 478

of both the C- and N- reserves and finally the release of soluble sugars in the growth 479

media The release of sugars to the media can be seen as a strategy to avoid a possible 480

inhibition of photosynthesis by negative sugar feedback (Oswald et al 2001) 481

CO2 supplementation did not promote increments in photosynthesis 482

Importantly photosynthesis in this study is defined as O2 release which is related to the 483


21

capacity to produce ATP and NADPH and not to CO2 assimilation Therefore we 484

cannot exclude a higher Calvin-Benson cycle activity in the presence of higher CO2 485

levels due to an inhibition of the photorespiratory pathway However it is highly 486

unlikely because we did not notice significant changes in cellular contents of glycine 487

and serine (Fig 7 Supplemental Tables S4 and S5) An increase in light intensity only 488

increased photosynthetic rates for cultures grown in the presence of ammonium and 489

urea and in fact HL treatment mostly led to a decrease in the amount of chlorophyll and 490

phycocyanin (Table II Supplemental Fig S2) The lower pigment content observed 491

under HL might lead to a lower redox state of the ETC (Alfonso et al 2000) which 492

might be a strategy to avoid production of reactive oxygen species and inhibition of 493

photosynthesis (Hihara et al 2001 Muramatsu et al 2009) particularly when 494

photoassimilates cannot be used for cell division Interestingly Kopečnaacute et al (2012) 495

showed that even with a lower cellular chlorophyll content under HL the rate of 496

chlorophyll synthesis was higher than at lower light intensity This was explained by a 497

reduction of the half-life of chlorophyll molecules and a greater demand for 498

chlorophylls as a result of faster growth rates 499

pII and ntcA are major proteins involved in sensing CN balance in 500

cyanobacteria (Flores and Herrero 2005) The expression levels of ntcA were not 501

affected by nitrogen sources as previously described (Alfonso et al 2001) pII and 502

ntcA transcript levels were positively correlated (rs = 083 Supplemental Fig S11) 503

which was expected because ntcA controls pII expression (Garciacutea-Domiacutenguez and 504

Florencio 1997 Lee et al 1999) ntcA and pII transcript levels correlated negatively 505

with total amino acids levels and positively with chlorophyll citrate isocitrate 506

fumarate malate and glutamate in agreement with their role in sensing CN balance 507

(Supplemental Fig S11) pII expression correlated positively with biomass (rs = 083 508


22

Fig 6) but not with Gt It is likely explained by the positive correlation between Gt and 509

biomass showing that cells dividing slowly accumulate more biomass and in particular 510

Carbon rich compounds Thus cells dividing slowly were not metabolically limited 511

however another factor clearly limits their growth 512

When Synechocystis was grown in the presence of urea it displayed fast division 513

rates one of the shortest growth cycles and lower metabolite contents (Fig 1 and 7 514

Table I) suggesting that urea may be a promising alternative to nitrate and ammonium 515

for growing cyanobacteria species of biotechnological interest In this condition cells 516

displayed higher photosynthetic rates when cultured under HL although it did not led to 517

increased growth rates (Tables I and II) These data reinforce our observations that 518

growth and photosynthesis are not directly linked Moreover even with a fast growth 519

rate and lower reserves cells may still present sink limitations to support the rate of 520

photoassimilates synthesis since the cellular levels of soluble sugars and their 521

concentrations in the media were similar to those observed under the other nine 522

treatments (Supplemental Fig S3A and S4) 523

Altogether our metabolic and physiological data indicate the absence of a 524

metabolic limitation of growth at log phase The photosynthetic rates observed were 525

high enough to support the synthesis of sugars and amino acids required for growth 526

which instead of being used for it were redirected for the synthesis of storage proteins 527

Indeed at day four of culture the protein content was positively correlated to Gt and 528

negatively with the number of cells suggesting that a major fraction of proteins were 529

used as nitrogen storage Moreover a significant amount of sugars were released to the 530

growth media instead of being used by the cellular metabolism 531

Is stationary phase reached because of metabolic nutrient limitation 532


23

Cultures under the different growth conditions reached stationary phase with 533

similar cell densities (Supplemental Table S1) despite of varying durations of culture 534

due to slightly different division rates (Fig 1 Table I) suggesting that a non-metabolic 535

factor is responsible for the onset of stationary phase In order to provide further support 536

for the above hypothesis we investigated the role of metabolism nutrient quorum 537

sensing and self-shading in the onset of stationary phase 538

At stationary phase the rate of photosynthesis remained high compared to those 539

observed at log phase decreasing by 50-60 under ML and being largely unaffected 540

under HL The decrease under ML is likely explained by self-shading and this 541

difference between HL and ML led to dramatic differences in the metabolic profiles of 542

cells grown under ML and HL The rate of photosynthesis correlated negatively with 543

glutamate at both ED and EN and positively with total soluble sugars (in cells and 544

culture media) proteins total soluble amino acids as well as the levels of some 545

individual amino acids such as leucine isoleucine alanine glycine and phenylalanine 546

at ED and proline at both ED and EN (Fig 8) Total soluble amino acids correlated 547

negatively with chlorophyll cyanophycin citrate isocitrate and glutamate and 548

positively with total soluble sugars (in cells and culture media) proteins and proline 549

(Fig 8) The negative correlations of amino acids with citrate isocitrate and glutamate 550

suggested that these metabolites had been used mainly as a source of carbon skeleton 551

for amino acids synthesis Additionally the correlations of total amino acids and soluble 552

sugars with proteins (rs = 07 and 079 respectively) and cyanophycin (rs = -082 and -553

062 respectively) indicate that protein synthesis was the main sink of these metabolites 554

(Fig 8 Supplemental Fig S12) Moreover positive correlations between arginine 555

proteins and biomass at this stage were observed (Fig 8 Supplemental Fig S12) 556


24

As the photoassimilates were not used for growth we observed an accumulation 557

of soluble sugars concomitant to an accumulation of amino acids and enhanced export 558

of sugars to the culture media (Fig 8) As a consequence of the higher export of sugars 559

to the media under HL than ML the levels of sucrose in cells under HL were much 560

lower than under ML (Supplemental Fig S8(B)) This disaccharide is described as a 561

compatible solute with no net charge and it is proposed that it can be accumulated 562

without interfering in the cellular homeostasis (Brown 1976 Mackay et al 1984 563

Mikkat et al 1997 Miao et al 2003 Hagemann 2011) Our data suggest that sucrose 564

might however have an effect on cellular homeostasis and in particular photosynthesis 565

possibly by sugar feedback inhibition of photosynthesis (Stitt et al 1991) Indeed in 566

contrast to ML cells grown under HL could maintain a high level of photosynthesis 567

while exporting larger amount of sucrose in the media and accumulating much lower 568

amount of sucrose within their cells Altogether the correlations suggest that stationary 569

phase was not achieved due to a limitation in the availability of building blocks andor 570

energy necessary for maintaining cell division 571

Considering that the metabolism is not controlling the rate of cell division we 572

tested whether already known factors were responsible such as (i) nutrient starvation 573

(Lazazzera 2000 Berla and Pakrasi 2012) (ii) quorum sensing (Sharif et al 2008) 574

and (iii) reduction of light quality and intensity available to the cells due to high cell 575

density (self-shading Raven and Kuumlbler 2002 Ort et al 2011 Lea-Smith et al 2014) 576

Nutrient deficiency could not account for the gradual decrease of cell division 577

rate because (1) a whole culture which had reached stationary phase did not re-start its 578

growth when transferred to a fresh media and (2) when we grew an inoculum into the 579

media obtained from a culture at stationary phase the inoculum could grow at the same 580

rate as in fresh media and an inhibition of growth was observed only if we attempted to 581


25

re-use a third time the media (Fig 3 Table III Supplemental Tables S8 and S9) For the 582

same reason low cell viability could not account for the decrease in cell division Our 583

conclusion differs from that of Schuurmans et al (2017) who concluded that the 584

transition to stationary phase is caused by nutrient limitation However the authors 585

artificially stopped growth by bubbling cultures with nitrogen gas (N2) which deprived 586

cells from CO2 and oxygen (O2) and indeed led to carbon starvation and growth arrest 587

However it does not mean a culture not deprived of CO2 and O2 would reach stationary 588

phase for the same reason 589

Quorum sensing is unlikely responsible because (1) an inoculum could grow 590

normally on a media obtained from a culture which had reached stationary phase (Table 591

III) and (2) an inoculum in a microtube fixed at bottom of a culture which had reached 592

stationary phase could grow almost normally under ML and normally under HL (Table 593

IV) This second observation also led us to conclude that light intensity and quality had 594

a minor impact on the onset of stationary phase Moreover cell density reached at 595

stationary phase was the same for 9 out of the 10 growth conditions we tested thus 596

independent from the two contrasting light intensities we applied (Fig 1 Supplemental 597

Table S1) Schuurmans et al (2017) showed that a gradual increase of light intensity 598

can lead cells to divide faster and avoid a linear phase Based on this observation the 599

authors affirmed that the decrease of cell division from log to linear phase is solely 600

caused by light limitation (self-shading) The authors used 30 micromol photons m-2 s-1 and 601

indeed cells might have been light limited thus leading to lower CO2 assimilation and 602

limitation of photoassimilates available for growth Moreover the authors stopped their 603

experiment after 72 hours by bubbling N2 which led to acute carbon starvation and 604

growth arrest while in our study we observed delayed growth due to cell density after 605

72-96 hours of growth Based on our results we suggest that cells can continue growing 606


26

after 72 hours and then will exhibit a gradual decrease of their growth rates reaching 607

linear phase before the onset of stationary phase irrespective of the light intensity 608

Our results indicate that cell density may affect negatively the growth rates of 609

cultures at relatively high densities as an artificial increase of density by 10 times led to 610

a decrease in the daily growth rate by 58 after 24 hours (plt0001) and 77 after 48 611

hours (plt00001 Fig 4C and D) Moreover cell density seems to be involved in the 612

control of the growth rates as demonstrated by fed-batch data Indeed we could stabilise 613

cell division over several days by maintaining cells at two given densities applying 614

different dilution rates Interestingly a dilution rate of 05 per day led to a division rate 615

of 05 time per day while a dilution rate of 2 times per day led to 2 divisions per day 616

(Fig 5C and D) which is close to the maximal division rates we could observe in our 617

batch cultures and in line with Kim et al (2011) Moreover when applying a dilution 618

rate of 2 times we first observed higher division rates (around 4 divisions per day) 619

which is close to those reported by Hihara et al (2001) and Zavřel et al (2015) This 620

fast growth rate led to an increase in the density of the culture before the division rates 621

stabilise and quantitatively counter-balance the daily dilution applied Altogether these 622

data suggest that cell density is involved in the control of the division rates in 623

Synechocystis 624

It is important to highlight that the observations in fed-batch cultures are not an 625

effect of fresh nutrients on growth since previous data indicate that media from a 626

cultures at stationary phase contain enough nutrients to support a second entire growth 627

cycle with similar growth rates (Fig 3) Even though self-shading showed moderate 628

effect at ML (Table IV) we propose that a cell-cell interaction process (Belas 2014 629

Ellison and Brun 2015) is involved in the control on cell division rate and that this 630

mechanism may be responsible for the gradual decrease in daily growth rates until the 631


27

onset of stationary phase when cells are cultured in batch At stationary phase cells can 632

still divide at very slow rates and reach higher densities Cultures maintained for 24 633

days after onset of stationary phase reached cell densities around 12 x 109 cell mL-1 634

(Fig 3) However during this period cells only divided 35 times 635

Even though our data suggest that a mechanism involving cell-cell interaction is 636

possibly involved in the control of division rates of Synechocystis when grown 637

planktonically this regulation seems absent when cells are grown on agar Indeed 638

Synechocystis normally forms dense colonies on agar exhibiting log growth and fast 639

doubling times of approximately 8 hours (Law et al 2000 Lamb et al 2014) This 640

observation suggests that Synechocystis growth regulation differs when cultured on agar 641

surface or in liquid Data from the literature show that heterotrophic bacteria exhibit 642

different gene expression patterns in biofilm and in planktonic growth (Imanaka et al 643

2010 He and Ahn 2011 Romero-Lastra et al 2017) Among the genes that are 644

upregulated under biofilm conditions some encode proteins located in the outer 645

membrane cell envelope and involved in transport or binding (Romero-Lastra et al 646

2017) Furthermore a significant number of the genes differentially expressed encode 647

for proteins of unknown functions (Imanaka et al 2010) Compared to heterotrophic 648

bacteria there is little information about biofilm formation in cyanobacteria (Parnasa et 649

al 2016) However recent studies have shown that Synechocystis and Synechococcus 650

elongatus PCC 7942 also exhibit variations in gene expression pattern when grown on 651

solid surface or in liquid (Nakasugi and Neilan 2005 Schatz et al 2013 Agostoni et 652

al 2016) Interestingly the Synechocystis strain used in the present study which is not 653

identified as ldquoglucose tolerantrdquo grew well in liquid BG11 supplemented with glucose 654

concentrations from 02 mM to 20 mM (Supplemental Fig S13) However as observed 655

by Williams (1988) colonies of the strain could not grow on agar containing more than 656


28

02 mM glucose (Supplemental Fig S13) It is thus possible that Synechocystis exhibits 657

variations in its regulation of growth when grown on solid or liquid media and that the 658

control of growth via cell-cell interaction we observed in liquid media does not occur on 659

solid media 660

This process of cell-cell interaction might be a strategy to avoid competition for 661

nutrients and light thus starvation and death If cells keep a constant rate of cell division 662

they would become limited by light and nutrients due to depletion of the nutrients in the 663

environment and self shading Thus they would die due to starvation since their 664

cellular stores are finite Hence a natural selection of individuals able to sense gradual 665

increase in density and regulate accordingly their division rates may have occurred 666

Besides cells which did not have this mechanism and exhibited an unrestricted growth 667

would be probably more susceptible to environmental variations as they would likely 668

have less stores to buffer environmental variations Our conclusions therefore 669

significantly vary from what is usually observed in non-photosynthetic bacteria where 670

starvation and quorum sensing are responsible for onset of stationary phase (Lazazzera 671

2000 Shiloach and Fass 2005) 672

CONCLUSION 673

Our data suggest that cell-cell interaction is possibly affecting growth and 674

promoting a gradual decrease of the division rate during the log phase As a 675

consequence the photoassimilates start to be stored instead of used for growth leading 676

to a decoupling between growth and photosynthesis This storage of metabolites 677

decreases the cellular sink capacity and cells start to export sugars to the media as an 678

alternative to avoid an inhibition of photosynthesis by negative feedback As the growth 679

rates in batch cultures seems to be primarily regulated by cell density dilutions are 680

necessary to maintain fast division rates for a long period Our conclusions have not 681


2

only biotechnological implications but also suggest that studies investigating major 682

drivers of the metabolic control of growth should be performed with care at culture 683

densities where a possible growth inhibition by cell density could occur ideally in fed-684

batch or continuous cultures in order to control precisely cell densities and then cell 685

division rates Another outcome of this study is that urea is a valuable nitrogen source 686

as we could get high division rates and short growth cycles This nitrogen source can be 687

an alternative to grow some cyanobacteria as an economically viable biomass 688

production since urea is cheaper than ammonium and nitrate its transport does not 689

require metabolic energy and during its assimilation there is a release of CO2 into to the 690

cell 691

692

Acknowledgements 693

AAE-F is recipient of Scholarships granted by the Agency for the Support and 694

Evaluation of Graduate Education (CAPES-Brazil ndashgrant BEX 890413-8) and the 695



(NUI Galway) 698

699

700

701

702

Tables 703

Table I Generation time of Synechocystis grown under ten culture conditions

Generation time ML HL

Nitrate 2141 plusmn 126 fg 2015 plusmn 163 efg

Ammonium 1695 plusmn 057 abcd 1930 plusmn 095 def


2

Urea 1488 plusmn 061 abc 1389 plusmn 020 a

Nitrate + CO2 2240 plusmn 017 g 1791 plusmn 037 cde

Ammonium + CO2 1744 plusmn 226 bcde 1470 plusmn 044 ab Generation time (hours) was calculated using a regression of the data within the logarithmic growth period at 50 micromol photons m-2 s-1 (ML) and 200 micromol photons m-2 s-1 (HL) with or without addition of CO2 Values represent the mean plusmn standard deviation (n = 3) Means followed by the same letter do not differ by 5 of probability (Tukey test)

704

Table II Photosynthetic rate in log and stationary phases Log Stationary ML HL ML HL

Nitrate 606 x10-10 plusmn 250 x10-11 def 544 x10-10 plusmn 800 x10-11 cde 228 x10-10 plusmn 374 x10-11 a 582 x10-10 plusmn 633 x10-11 b

Ammonium 461 x10-10 plusmn 618 x10-11 abcd 723 x10-10 plusmn 469 x10-11 f 275 x10-10 plusmn 241 x10-11 a 555 x10-10 plusmn 523 x10-11 b

Urea 389 x10-10 plusmn 292 x10-11 ab 620 x10-10 plusmn 756 x10-11 ef 223 x10-10 plusmn 963 x10-12 a 370 x10-10 plusmn 111 x10-10 a

Nitrate + CO2 529 x10-10 plusmn 292 x10-11 bcde 572 x10-10 plusmn 741 x10-11 def 315 x10-10 plusmn 782 x10-12 a 609 x10-10 plusmn 110 x10-11 b

Ammonium + CO2 401 x10-10 plusmn 247 x10-11 abc 318 x10-10 plusmn 364 x10-11 a 259 x10-10 plusmn 136 x10-11 a 302 x10-10 plusmn 666 x10-11 a

Photosynthetic rate (micromol O2 Lscell) determined in the fourth day of culture (log phase) and after three days in the stationary phase at 50 micromol photons m-2 s-1 (ML) and 200 micromol photons m-2 s-1 (HL) with or without addition of CO2 Statistics as described for table 1

705

706

707

708

709

710

711

712

713

714

Table III Ratio between cell density at day 4 (D4) and day 0 (D0) of (1) an

inoculum grown on filtered media from a previous culture which had reached

stationary phase or (2) whole cell culture which had reached stationary phase

and was re-grown without dilution on fresh media

1 Control

Filtered media inoculated with 2 (vv)

cells ML HL ML HL


3

Nitrate 109 plusmn 11 197 plusmn 18 133 plusmn 36 144 plusmn 54

Ammonium 167 plusmn 04 155 plusmn 19 174 plusmn 34 119 plusmn 18

Urea 189 plusmn 04 204 plusmn 13 229 plusmn 61 17 8 plusmn 19

Nitrate + CO2 195 plusmn 04 167 plusmn 23 146 plusmn 35 182 plusmn 04

Ammonium + CO2 205 plusmn 42 175 plusmn 11 147 plusmn 20 190 plusmn 66

2 Control Cell pellet re-grown on

fresh media ML HL ML HL



Urea 13 plusmn 01 18 plusmn 01 14 plusmn 03 16 plusmn 02


Ammonium + CO2 12 plusmn 01 18 plusmn 01 15 plusmn 02 17 plusmn 01 Ratio D4D0 under 50 micromol photons m-2 s-1 (ML) and 200 micromol photons m-2 s-1 (HL) with or without addition of CO2 Control ratios were calculated from data obtain on growth curves showed on figure 1 (1) Between the first 4 days of culturing and (2) the last four days of culturing Values represent the mean plusmn standard deviation (n = 3) Asterisk represents significant difference between control and treatment for the same growth condition (plt005 T-test) Cell densities for each growth condition and treatment are present in Supplemental tables S8 and S9

715

716

717

718

719

720

721

722

723

Table IV Cell density of an inoculum grown in microtubes fixed at the bottom

of flask containing BG11 fresh media (control) or a culture which had reached

stationary phase (treatment)

Urea ndash ML

Day 0 Day 4 Ratio (D4D

0)

Control 569x106 plusmn 763x10

5 431x10

7 plusmn 607x10

6 758 plusmn 039

Treatment 523x106 plusmn 819x10

5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162

Cell number (cell numbermL-1) of urea cultures grown under 50 micromol photons m-2 s-1 (ML) and 200 micromol photons m-2 s-1 (HL) Values represent the mean plusmn standard deviation (n = 3) Asterisk represents significant difference between means for the same growth condition (plt005 Paired T-test)

724

Figure legends 725

Figure 1 Synechocystis growth cycle represented on a Log(cell) basis (A C and E) and daily number of 726

division (B D and F) when grown on ten different culture conditions Cultures on nitrate (A-B) 727

ammonium (C-D) and urea (E-F) were submitted to two different light intensities 50 micromol photons m-2 s-728

1 (red) and 200 micromol photons m-2 s-1 (blue) Cultures grown on ammonium and nitrate were supplemented 729

with 25 mM bicarbonate (triangles) or not supplemented (circles) 730

731

Figure 2 Experimental setup for the identification of the processes involved in the onset of stationary 732

phase 733

734

Figure 3 Growth cycles of Synechocystis on fresh BG-11 (blue circles) old BG-11 (brown circle) and 735

reused old BG-11 (pink circles) Red arrows show when the entire culture was centrifuged and the 736

supernatant was filtered and used to grow an inoculum obtained from the resuspended pellet during 737

additional 8 days Black arrow shows when cultures with reused old BG-11 were divided in two parts and 738

supplemented in a proportion of 11 with fresh BG-11 (yellow circles) or water (red triangles) As a 739

control cultures we maintained growing as batch for the entire period of 32 days (green squares) 740

741

Figure 4 Experimental setups to test the effect of cell density on Synechocystis growth Effect of 742

different initial cell densities on growth cycle (A) and daily number of divisions (B) 2 x 106 (black) 4 x 743

106 (red) 6 x 106 (green) and 2 x 107 cells mL-1 (yellow) Effect of an artificial increase of cell density on 744

growth cycle (C) and daily number of divisions (D) Red arrow in C indicated artificial increase of cell 745

density Cultures control (black) were not disturbed during their growth Control centrifugation (red) were 746


5

submitted to the same pipetting centrifuging and growth conditions of 10x concentration but not 747

concentrated after centrifugation 10x concentration (green) were artificially concentrated after 748

centrifugation Asterisk represents significant differences from 2 x 106 cells mL-1 (B) or control (D) 749

(plt005 t-test) 750

751

Figure 5 Experimental setups to test the effect of cell density on Synechocystis growth Growth cycle 752

and daily number of divisions in cultures grown in fed-batch condition starting at low cell density (A-B) 753

and high cell density (C-D) Red arrow (A) indicates that cultures were diluted to the same initial density 754

of day 0 755

756

Figure 6 Spearmanrsquos rank coefficient of correlation between Gt Biomass photosynthetic rate (A) and 757

cell number and metabolites and gene expression levels determined at the end of the day and end of the 758

night in the fourth day of culture Total soluble sugars (media) was determined only at the end of the 759

night Significant correlations at 5 of probability are highlighted in green (positive) and red (negative) 760

Additional correlations between all growth physiologic metabolic and molecular traits are present in 761

figure S11 762

763

Figure 7 Heatmap of metabolites levels Culture conditions are ordered based on growth rates from the 764

fastest (left) to the slowest (right) A- ammonium N- nitrate U- urea C ndash CO2 50 - 50 micromol photons m-2 765

s-1 200- 200 micromol photons m-2 s-1 766

767

Figure 8 Spearmanrsquos rank coefficient of correlation between Biomass photosynthetic rate (A) and total 768

amino acids and metabolites levels determined at the end of the day and end of the night in cells three 769

days after onset of the stationary phase Total soluble sugars (media) was determined only at the end of 770

the night Significant correlations at 5 of probability are highlighted in green (positive) and red 771

(negative) Additional correlations between all growth physiologic metabolic and molecular traits are 772

present in figure S12 773

774

SUPPLEMENTAL DATA 775


6

Supplemental Figure S1 Relative expression levels of ftsZ at the end of the day and at the end of the 776

night in the fourth day of culture and three days after onset of the stationary phase 777

Supplemental Figure S2 Chlorophyll a and phycocyanin levels determined in the fourth day of culture 778

Supplemental Figure S3 Total soluble sugars glycogen total soluble amino acids proteins and 779

cyanophycin levels determined in the fourth day of culture at the end of the day and the end of the night 780

Supplemental Figure S4 Total soluble sugars concentrations in the growth media after four days of 781

culture Samples were harvested at the end of the night 782

Supplemental Figure S5 Percentage of glycogen degraded during the night of the fourth day of culture 783

Supplemental Figure S6 Relative expression levels of ntcA and pII at the end of the day and end of the 784

night of the fourth day of culture 785

Supplemental Figure S7 Chlorophyll a and phycocyanin levels three days after onset of stationary 786

phase 787

Supplemental Figure S8 Total soluble sugars sucrose and glycogen levels three days after onset of 788

stationary phase 789

Supplemental Figure S9 Total soluble sugars concentrations in the growth media three days after onset 790

of stationary phase 791

Supplemental Figure S10 Total soluble amino acids glutamate cyanophycin and proteins contents in 792

cells three days after onset of the stationary phase 793

Supplemental Figure S11 Spearmanrsquos rank coefficient of correlation between all growth physiological 794

metabolic and molecular traits determined at the end of the day and end of the night in the fourth day of 795

culture 796


and metabolic traits determined at the end of the day and end of the night in cells three days after onset of 798

the stationary phase 799

Supplemental Figure S13 Glucose tolerance test Synechocystis sp PCC 6803 cultured in mixotrophic 800

conditions in liquid and agar BG11 supplemented with nitrate and different glucose concentrations 801

Supplemental Table S1 Cell number after four days of culture (log phase) and three days after onset of 802 the stationary phase 803

Supplemental Table S2 Cell size in log and stationary phases 804

Supplemental Table S3 Cellular dry weight in log and stationary phases 805


7

Supplemental Table S4 Primary metabolites determined by gas chromatography coupled with mass 806 spectrometry (GC-MS) at the end of the day in the fourth day of culture 807 Supplemental Table S5 Primary metabolites determined by gas chromatography coupled with mass 808 spectrometry (GC-MS) at the end of the night in the fourth day of culture 809 Supplemental Table S6 Primary metabolites determined by gas chromatography coupled with mass 810 spectrometry (GC-MS) at the end of the day of the third day after onset of stationary phase 811 Supplemental Table S7 Primary metabolites determined by gas chromatography coupled with mass 812 spectrometry (GC-MS) at the end of the night of the third day after onset of stationary phase 813 Supplemental Table S8 Cell density of an inoculum grown on filtered media from a previous culture 814 which had reached stationary phase or whole cell culture which had reached stationary phase and was re-815 grown without dilution on fresh media 816 Supplemental Table S9 Cell density of an inoculum grown on filtered media from a previous culture 817 which had reached stationary phase or whole cell culture which had reached stationary phase and was re-818 grown without dilution on fresh media 819 820


1

821


1

Figure 1 Synechocystis growth cycle represented on a Log2(cell) basis (A C and E) and daily number of division (B D and F) when grown on ten different culture conditions Cultures on nitrate (A-B) ammonium (C-D) and urea (E-F) were submitted to two different light intensities 50 micromol photons m-2 s-1 (red) and 200 micromol photons m-2 s-1 (blue) Cultures grown on ammonium and nitrate were supplemented with 25 mM bicarbonate (triangles) or not supplemented (circles)


2

Figure 2 Experimental setup for the identification of the processes involved in the onset of stationary phase


3

Figure 3 Growth cycles of Synechocystis on fresh BG-11 (blue circles) old BG-11 (brown circle) and reused old BG-11 (pink circles) Red arrows show when the entire culture was centrifuged and the supernatant was filtered and used to grow an inoculum obtained from the resuspended pellet during additional 8 days Black arrow shows when cultures with reused old BG-11 were divided in two parts and supplemented in a proportion of 11 with fresh BG-11 (yellow circles) or water (red triangles) As a control cultures we maintained growing as batch for the entire period of 32 days (green squares)


4

Figure 4 Experimental setups to test the effect of cell density on Synechocystis growth Effect of different initial cell densities on growth cycle (A) and daily number of divisions (B) 2 x 106 (black) 4 x 106 (red) 6 x 106 (green) and 2 x 107 cells mL-1 (yellow) Effect of an artificial increase of cell density on growth cycle (C) and daily number of divisions (D) Red arrow in C indicated artificial increase of cell density Cultures control (black) were not disturbed during their growth Control centrifugation (red) were submitted to the same pipetting centrifuging and growth conditions of 10x concentration but not concentrated after centrifugation 10x concentration (green) were artificially concentrated after centrifugation Asterisk represents significant differences from 2 x 106 cells mL-1 (B) or control (D) (plt005 t-test)


5

Figure 5 Experimental setups to test the effect of cell density on Synechocystis growth Growth cycle and daily number of divisions in cultures grown in fed-batch condition starting at low cell density (A-B) and high cell density (C-D) Red arrow (A) indicates that cultures were diluted to the same initial density of day 0


6

Figure 6 Spearmanrsquos rank coefficient of correlation between Gt Biomass photosynthetic rate (A) and cell number and metabolites and gene expression levels determined at the end of the day and end of the night in the fourth day of culture Total soluble sugars (media) was determined only at the end of the night Significant correlations at 5 of probability are highlighted in green (positive) and red (negative) Additional correlations between all growth physiologic metabolic and molecular traits are present in figure S11


7

Figure 7 Heatmap of metabolites levels Culture conditions are ordered based on growth rates from the fastest (left) to the slowest (right) A- ammonium N- nitrate U- urea C ndash CO2 50 - 50 micromol photons m-2 s-1 200- 200 micromol photons m-2 s-1


8

Figure 8 Spearmanrsquos rank coefficient of correlation between Biomass photosynthetic rate (A) and total amino acids and metabolites levels determined at the end of the day and end of the night in cells three days after onset of the stationary phase Total soluble sugars (media) was determined only at the end of the night Significant correlations at 5 of probability are highlighted in green (positive) and red (negative) Additional correlations between all growth physiologic and metabolic traits are present in figure S12


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Parsed Citations

Reviewer PDF

Figures

Parsed Citations

2

Funding information 31 AAE-F is recipient of Scholarships granted by the Agency for the Support and 32

Evaluation of Graduate Education (CAPES-Brazil ndash grant BEX 890413-8) and the 33



(NUI Galway) 36

37 Corresponding author Dr Ronan Sulpice Tel +353-(0)-91494292 Email 38

ronansulpicenuigalwayie 39

40 41 42

43


1

ABSTRACT 44

















1

INTRODUCTION 60









et al 2016) 69

















2






















value compounds 106





3

















126


4



























5



period 154
























6



















before 195








7




















Dry Biomass 221







8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

1

ABSTRACT 44

















1

INTRODUCTION 60









et al 2016) 69

















2






















value compounds 106





3

















126


4



























5



period 154
























6



















before 195








7




















Dry Biomass 221







8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

1

INTRODUCTION 60









et al 2016) 69

















2






















value compounds 106





3

















126


4



























5



period 154
























6



















before 195








7




















Dry Biomass 221







8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

2






















value compounds 106





3

















126


4



























5



period 154
























6



















before 195








7




















Dry Biomass 221







8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

3

















126


4



























5



period 154
























6



















before 195








7




















Dry Biomass 221







8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

4



























5



period 154
























6



















before 195








7




















Dry Biomass 221







8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

5



period 154
























6



















before 195








7




















Dry Biomass 221







8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

6



















before 195








7




















Dry Biomass 221







8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

7




















Dry Biomass 221







8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

8



























9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

9

























276


10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

10

277


11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

11

RESULTS 278


























12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

12



























13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

13























Fig S3(E)) 350




14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

14



























15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

15



























16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

16


























17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

17


(Fig 4A and B) 428







and D) 435


















18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

18





cultures 456

457


19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

19

458


20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

20

DISCUSSION 459


























21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

21



























22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

22


























23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

23


























24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

24



























25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

25



























26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

26


















Synechocystis 624









27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

27



























28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

28




solid media 660













CONCLUSION 673










2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

2










cell 691

692






(NUI Galway) 698

699

700

701

702

Tables 703






2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

2




704








705

706

707

708

709

710

711

712

713

714





1 Control


cells ML HL ML HL


3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

3













715

716

717

718

719

720

721

722

723




Urea ndash ML


0)


5 431x10

7 plusmn 607x10

6 758 plusmn 039


5 243x10

7 plusmn 286x10

6 467 plusmn 033


4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

4

Urea ndash HL


0)


5 381x10

7 plusmn 136x10

7 1100 plusmn 123


5 340x10

7 plusmn 790x10

6 1083 plusmn 162


724

Figure legends 725






731


phase 733

734







741







5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

5




(plt005 t-test) 750

751




of day 0 755

756






figure S11 762

763




767







774



6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

6












phase 787









culture 796










7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

7



1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

1

821


1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

1



2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

2



3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

3



4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

4



5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

5



6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

6



7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

7



8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

8





















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations



















































































































































































Parsed Citations

Reviewer PDF

Figures

Parsed Citations

A novel mechanism, linked to cell density, largely ... · 1 1 A novel mechanism, linked to cell...

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Transcript of A novel mechanism, linked to cell density, largely ... · 1 1 A novel mechanism, linked to cell...