Identification of Optimal Conditions for Growth and Pigment Production in indegenous Dunaliella salina strain Using Central Composite Design Method

Abstract

Microalgae are widely used in biotechnological applications due to their natural compounds such as protein, lipid and colored pigments. Dunaliella salina, a green algae tolerant to high salt concentrations, is a natural source of beta-carotene, and therefore it is one of the microalgae species that is prominent in biotechnological productions. Beta-carotene accumulation occurs in D. salina cells under stress conditions such as high salinity, high temperature, intense light or nutrient scarcity, but these conditions also negatively affects the cell growth. A two-phase production system is usually applied to the cultures in which the cell growth is first promoted and then the pigment production is increased by applying the stress condition. In this study, it was aimed to determine the culture conditions that would simultaneously promote biomass growth and intracellular carotenoid accumulation without the need for two phase production by using the central composite design method for a Dunaliella salina strain isolated from local sources. Two media components that cause carotenoid accumulation in the cell; salt (NaCl) and nitrate (KNO3) were selected as the factors to be used for experimental design. As a result of the experiments carried out at three different levels of factors, it was determined that the amount of salt in the medium was the primary factor in terms of both biomass increase and carotenoid accumulation. For D. salina cells, culture medium containing 1.75 M NaCl and 1.2 mM KNO3 was determined as the optimum condition for both increasing the amount of biomass and pigment accumulation in parallel. Carotenoid deposition was significantly affected by salt concentration, while the amount of nitrate used in the media was found to have a lower effect in the selected range.

Keywords

• Biomass
• Carotenoids
• Central Composite Design
• Dunaliella salina

Yerli Dunaliella salina suşunda Büyüme ve Pigment Üretimi için Optimal Koşulların Merkezi Kompozit Tasarım Yöntemi Kullanılarak Belirlenmesi

Öz

Mikroalgler, sahip oldukları protein, lipid ve renkli pigmentler gibi doğal bileşikler sebebi ile biyoteknolojik uygulamalarda geniş bir kullanım alanı bulmaktadır. Yeşil algler sınıfına dahil olan ve yüksek tuz toleransı gösterebilen Dunaliella salina türü, doğal bir beta-karoten kaynağı olması sebebi ile biyoteknolojik üretimlerde öne çıkan mikroalg türleri arasındadır. Yüksek tuzluluk, yüksek sıcaklık, yoğun ışık miktarı ya da besin kıtlığı gibi stres koşulları altında D. salina hücrelerinde beta-karoten birikimi gerçekleşmekte, ancak bu durum hücre büyümesini olumsuz etkilemektedir. Bu sebeple genellikle hazırlanan kültürlerde önce hücre büyümesinin teşvik edildiği, ardından stres koşulu uygulanarak pigment üretiminin artırıldığı iki fazlı üretim sistemi benimsenmiştir. Bu çalışmada yerel kaynaklardan izole edilen bir D. salina suşu için merkezi kompozit tasarım yönteminden faydalanılarak biyokütle artışı ve hücre içi karotenoid birikimini, iki fazlı üretime gerek olmaksızın, eş zamanlı olarak teşvik edecek ortam koşullarının belirlenmesi amaçlanmıştır. Hücre içinde karotenoid birikimine sebep olan iki ortam bileşeni; tuz (NaCl) ve nitrat (KNO3), deney tasarımı için kullanılacak faktörler olarak seçilmiştir. Faktörlerin üç farklı düzeyinde gerçekleştirilen denemeler sonucunda ortamdaki tuz miktarının hem biyokütle artışı hem de karotenoid birikimi açısından birincil etmen olduğu belirlenmiştir. D. salina hücreleri için 1,75 M NaCl ve 1,2 mM KNO3 içeren kültür ortamında biyokütlenin, ve paralel olarak hücre içi pigment miktarının artışı için optimum koşul olarak belirlenmiştir. Karotenoid birikimi, tuz konsantrasyonu açısından önemli ölçüde etkilenirken, kullanılan nitrat miktarının seçilen aralıkta daha düşük etki gösterdiği görülmüştür.

Anahtar Kelimeler

• Biyokütle
• Karotenoidler
• Merkezi Kompozit Tasarım
• Dunaliella salina

References

1. Benavente-Valdés J.R., Aguilar C., Contreras-Esquivel J.C., Méndez-Zavala A., Montañez A., (2016). Strategies to enhance the production of photosynthetic pigments and lipids in chlorophycae species. Biotechnology Reports, 10: 117-125 DOI: 10.1016/j.btre.2016.04.001
2. Borowitzka, M. A. (1988). Algal growth media and sources of cultures, In: Borowitzka M.A. ve Borowitzka L.J. (eds.), Micro-algal Biotechnology. Cambridge University Press: Cambridge. pp. 456-465, https://doi.org/10.1002/jctb.280470214
3. Borowitzka M.A. (1988). Vitamins and fine chemicals. In: Borowitzka M.A. ve Borowitzka L.J. (eds), Micro-algal Biotechnology. CambridgeUniversity Press, Cambridge, 153-196. https://doi.org/10.1002/jctb.280470214
4. Chen C.Y., Lu I.C., Nagarajan D., Chang C.H., Ng I.S., Lee D.J., Chang J.S. 2018. A highly efficient two-stage cultivation strategy for lutein production using heterotrophic culture of Chlorella sorokiniana MB-1-M12. Bioresource Technology, Volume 253: 141-147. https://doi.org/10.1016/j.biortech.2018.01.027
5. Chen H. ve Jiang J.G., 2009. Osmotic responses of Dunaliella to the changes of salinity. J Cell Physiol. 219(2):251-8. DOI: 10.1002/jcp.21715
6. Çelekli A. ve Dönmez G. (2006). Effect of pH, light intensity, salt and nitrogen concentrations on growth and b-carotene accumulation by a new isolate of Dunaliella sp. World Journal of Microbiology & Biotechnology. 22: 183–189. DOI: 10.1007/s11274-005-9017-0
7. Değirmencioğlu A. ve Yazgı A. (2006). Tepki Yüzeyleri Metodolojisi "Optimizasyon Esaslı Çalışmalara İlişkin Teorik Esaslar ve Tarımsal Mekanizasyon Uygulamaları" . Tarım Makinaları Bilimi Dergisi 2(2): 111-115.
8. Del Campo J.A., Garcia-Gonzales M., Guerrero M.G. (2007). Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol 74: 1163-1174. DOI: 10.1007/s00253-007-0844-9

9. Diler, İ., Dilek, K., 2002. Significance of Pigmentation and Use in Aquaculture. Turkish Journal of Fisheries and Aquatic Sciences 2: 97-99
10. Fujisawa, M., Takita, E., Harada, H., Sakurai, N., Suzuki, H., Ohyama,K., Shibata, D., Misawa, N., 2009. Pathway engineering of Brassica napus seeds using multiplekey enzyme genes involved in ketocarotenoid formation. J Exp Bot, 60(4):1319-32. DOI: 10.1093/jxb/erp006.
11. Guerin M., Huntley M.E., Olaizola M. (2003). Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 21:210–216. https://doi.org/10.1016/S0167-7799(03)00078-7
12. Hosseini Tafreshi A. ve Shariati M. (2009). Dunaliella biotechnology: methods and applications. Journal of Applied Microbiology, 107: 14–35. https://doi.org/10.1111/j.1365-2672.2009.04153.x
13. Jin E.S. ve Melis A. (2003). Microalgal Biotechnology: Carotenoid production by the green algae Dunaliella salina. Biotechnology and Bioprocess Engineering. 8: 331-337. DOI: 10.1007/BF02949276
14. Johnson, M.K., Johnson, E.J, Mac Elroy, R.D., Speer, H.L. and Bruff, B.S. (1968). Effects of salts on the halophilic alga Dunaliella viridis. J. Bacteriology 95: 1461-1468.
15. Khan M.I., Shin J.H., Kim J.D. (2018). The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microb. Cell Fact. 17:36 https://doi.org/10.1186/s12934-018-0879-x
16. Khuri A.I., (2017). A General Overview of Response Surface Methodology. Biom Biostat Int J 5(3): 00133. DOI: 10.15406/bbij.2017.05.00133
17. Lichtenthaler HK, Buschmann C (2001). Chlorophylls and Carotenoids: Measurement and Characterization by UV‐VIS Spectroscopy. In: Current Protocols in Food Analytical Chemistry. New York, NY, USA: John Wiley and Sons, Inc. F4.3.1-F4.3.8. https://doi.org/10.1002/0471142913.faf0403s01
18. Marin N., Morales F., Lodeiros C., Tamigneaux E. (1998). Effect of nitrate concentration on growth and pigment synthesis of Dunaliella salina cultivated under low illumination and preadapted to different salinities. Journal of Applied Phycology 10: 405–411, 1998. DOI: 10.1023/A:1008017928651
19. Morowvat M.H. ve Ghasemi Y. (2016). Culture medium optimization for enhanced β-carotene and biomass production by Dunaliella salina in mixotrophic culture Biocatalysis and Agricultural Biotechnology 7: 217–223. https://doi.org/10.1016/j.bcab.2016.06.008
20. Oren A., 2014. The ecology of Dunaliella in high-salt environments. Journal of Biological Research-Thessaloniki , 21:23, DOI: 10.1186/s40709-014-0023-y
21. Osanai T., Park Y.I., Yuki Nakamura Y. (2017). Editorial: Biotechnology of Microalgae, Based on Molecular Biology and Biochemistry of Eukaryotic Algae and Cyanobacteria. Front. Microbiol. 8:118. DOI: 10.3389/fmicb.2017.00118
22. Pisal D.S. ve Lele S.S. (2005). Carotenoid production from microalga, Dunaliella salina. Indian Journal of Biotechnology 4: 476-483.
23. Pulz, O. ve Gross, W., 2004. Valuable products from biotechnology of microalgae. Appl. Microbiol. Biotechnol. 65(6): 635-648. DOI: 10.1007/s00253-004-1647-x