Neha Sachdeva Université De Mons, Belgium Changes in chemistry of N sources induce changes in growth and physiology of Arthrospira sp. PCC 8005 Neha Sachdeva Université De Mons, Belgium Co-authors Deschoenmaeker F., Bayon-Vicente G., Depraetere O., Cabrera Pino Juan C., Leroy B., Muylaert K., Wattiez R.

MELISSA LOOP Urine NH4+

STUDY DESIGN NH4+ NO3- Urea Growth profile N Chemistry Proteomics

Batch Culture Results

No cell growth at 12mM [NH4+]. Growth Profile Longer lag phase for 6mM NH4 , known to be at inhibitory concentration, more biomass lesser nh4 available, no pH control [NO3-] [NH4+] [Urea] No cell growth at 12mM [NH4+].

Fastest assimilation of Urea: 2mM & 6mM N Metabolism [NO3-] [NH4+] [Urea] Fastest assimilation of Urea: 2mM & 6mM

Urea is a better N source for Arthrospira sp. PCC 8005 cultivation. N Uptake Rate [NO3-] [NH4+] [Urea] 2mM & 6mM : Highest Uptake rate: Urea 12mM: Comparable uptake rate for Urea and nitrate Urea is a better N source for Arthrospira sp. PCC 8005 cultivation. 𝑁 𝑢𝑝𝑡𝑎𝑘𝑒 𝑟𝑎𝑡𝑒(𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 𝑎𝑡 𝑒𝑥ℎ𝑎𝑢𝑡𝑖𝑜𝑛 𝑜𝑓 𝑁 𝑠𝑜𝑢𝑟𝑐𝑒)= 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑁𝑡𝑜𝑡𝑎𝑙 (𝑚𝑀) 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑚𝑔 ∗𝑡𝑖𝑚𝑒(ℎ𝑟)

Arthropira sp. PCC 8005 Nitrogen Metabolism NO3- (/ NO2-) NH4+ Urea NO3- Nar Gln Glu GS GOGAT Ur CO2 NtcA , … 2-OG NO2- Nir Cell Membrane Nitrate (NO3-) predominate in environment. Ammonium (NH4+) preferred source according to its redox state. Urea/[NO3-]/Nitrite (NO2-) reduced to simpler [NH4+]. [NH4+] reversibly inhibits assimilation of other N sources. Inhibition caused due to repression of: Transmembrane transporter: NrtABCD and NrtP N Assimilation Enzymes: Nar (NO3-) and Nir (NO2-). GS-GOGAT Cycle [NH4+] enters the GS-GOGAT cycle. Glutamine synthetase (GS) catalyses an ATP-dependent amination of glutamate to yield glutamine. Glutamine oxoglutarate aminotransferase (GOGAT) catalyses the reductive transfer of the amide group from glutamine to 2-oxoglutarate (2-OG) leading to the production of 2 glutamates. Regulation felicitated by transcription factors NtcA, which in turn regulates genes involved in N metabolism.

Transcriptomic Analysis 20mM [NO3-] 4mM [NH4+] + A RTPCR MS/MS SWATH Analysis 20mM [NO3-] 2mM Urea + 2mM Urea B

Transcript Relative Abundance NO3 & NH4 20mM [NO3-] 20mM [NO3-] + 4mM [NH4+] 4mM[NH4+] [NH4+] presence inhibits expression of genes for [NO3-] metabolism De-repression of genes under N depletion Lochab et al., 2014

Transcript Relative Abundance NO3 & Urea 20mM [NO3-] 20mM [NO3-] + 2mM Urea 2mM Urea Urea presence inhibits expression of genes for [NO3-] metabolism De-repression of genes under N depletion Lochab et al., 2014

PBR Culture 28mM NO3- 28mM NH4+

PBR Growth Profile First 5 days: Batch mode28mM [NO3-] . Turbidostat mode on: Day 7 First transit to 28mM [NH4+] on Day 13; Second transit to 28mM [NO3-] on Day 30.

Cyanophycin Profile Indicative of N depletion. Cyanophycin (CP) : N storage unit. Increased under [NO3-]; Decreased under [NH4+]. Indicative of N depletion. Could be corroborated with proteomic analysis

Lipids Profile No significant variation in Total Lipid content Eicosane shown to decrease under presence of [NH4+] & recovered in [NO3-] Indicative of nutrient stress* Similar trends (though minimal) observed for Palmatic acid & Linoleic acid. *Ying Yang, 2013

Exopolysaccarides Significant increase in glycerol pyranoside. Glycerol known to play in role in osmotic acclimatization Cell wall synthesis :10.63 fold increase seen in enzyme UDP-dehydrogenase Indicative: Cellular stress Hagemann, 2011 Katpaka & Misaki, 1983 Weiwer et al.., 2008

Conclusions Urea indicated as better N source Highest N uptake and assimilation rate Presence of [NH4+] induced repression of [NO3-] assimilation pathways and genes Similar trend in presence of Urea (end of 10 hrs) due to potential accumulation of [NH4+]: Urea Hydrolysis Arthrospira sp. could be cultivated at 8.5 mM [NH4+]; at controlled pH (lower than pka 9.25). Inhibitory & N depletion effect of [NH4+] could be avoided by pulse feeding (after exhaustion)*. *Rodrigues et al., 2010

ACKNOWLEGMENTS Deschoenmaeker F.1, Bayon-Vicente G.1, Depraetere O.2, Sachdeva N.1, Cabrera Pino Juan C.3, Leroy B.1, Muylaert K.2 and Wattiez R.1 1Department of Proteomic and Microbiology, Research Institute for Biosciences, University of Mons, Place du Parc 20, B-7000 Mons, Belgium. 2Laboratory Aquatic Biology, KU Leuven campus Kortrijk, E. Sabbelaan 53, 8500, Kortrijk, Belgium 3Biotechnology unit, Materia Nova, Rue des Foudriers 1, B-7822 Ghislenghien, Belgium.

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