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Chlorella sorokiniana’ nın İzolasyonu, Moleküler Tanılanması, Fototrofik, Miksotrofik ve Heterotrofik Üretimi

Yıl 2021, Cilt: 7 Sayı: 2, 128 - 137, 26.08.2021

Döndü Yalçın Bingül , Zeliha Demirel , Meltem Conk Dalay

https://doi.org/10.17216/limnofish.703234

Öz

İzmir’in Gümüldür bölgesinden alınan su örneğinde, seyreltme ve dökme plaka yöntemleri kullanılarak mikroalg izolasyonu yapılmıştır. Işık mikroskobuyla morfolojik olarak değerlendirilen türün Chlorella sp. olduğu saptanmıştır. Moleküler yöntemlerle mikroalg DNA’sı izole edilerek 16S ve 18S rRNA gen bölgeleri PCR’da çoğaltılmıştır. Bu dizinin sekanslanması ve filogenetik olarak değerlendirilmesi sonucu Chlorella sorokiniana olduğu belirlenmiştir. Aksenik C. sorokiniana elde etmek için santrifüj ile yıkama, antibiyotik ile muamele, agar ortamında büyütme ve tek hücre izolasyonu gibi farklı yöntemler kullanılarak aksenikleştirme işleminden başarılı sonuçlar elde edilmiştir. Fototrofik C. sorokiniana’ dan elde edilen biyokütle (0,19 g L-1) ve spesifik büyüme hızı (0,78 gün-1), miksotrofik C. sorokiniana’ dan elde edilen biyokütle (0,31 g L-1) ve spesifik büyüme hızı(1,3 gün-1), heterotrofik C. sorokiniana’ dan elde edilen biyokütle (0,6 g L-1) ve spesifik büyüme hızı (2,52 gün-1) olarak belirlenmiştir. Elde edilen bulgulara göre aksenik mikroalg C. sorokiniana’ nın farklı üretim koşullarındaki biyokütle verimliliği şu şeklide sıralanabilir: heterotrofi>miksotrofi>fototrofi.

Anahtar Kelimeler

Chlorella sorokiniana moleküler tanılama Fototrofi Miksotrofi Heterotrofi

Teşekkür

Sevgili Doç. Dr. Müge İşleten Hoşoğlu'na katkılarından dolayı teşekkürler.

Kaynakça

  • Abreu AP, Fernandes B, Vicente AA, Teixeira J, Dragone G. 2012. Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source. Bioresour Technol. 118:61-66. doi: 10.1016/j.biortech.2012.05.055
  • AlgaeBase. 2021. [Erişim tarihi: 09 Mar 2021]. Erişim Adresi: https://www.algaebase.org/search/species/detail/?species_id=r7b5b5e3003154f99
  • AlgaePARC. 2021. Wageningen University&Research, [Erişim tarihi: 09 Mar 2021]. Erişim Adresi: http://www.algaeparc.com/news
  • Baytut Ö, Gürkanli CT, Gönülol A, Özkoç I. 2014. Molecular phylogeny of Chlorella-related chlorophytes (Chlorophyta) from Anatolian freshwaters of Turkey. Turkish Journal of Botany. 38(3): 600-607. doi:10.3906/bot-1304-32
  • Becker EW. 1995. Microalgae biotechnology and microbiology. Cambridge University Press. p. 293. doi.org/10.1002/jctb.280470214
  • Becker EW. 2007. Microalgae as a source of protein. Biotechnol Adv. 25(2):207-210. doi: 10.1016/j.biotechadv.2006.11.002
  • BLAST. 2021. [Erişim tarihi: 09 Mar 2021]. Erişim Adresi:https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_395406506
  • Borowitzka MA. 1992. Algal biotechnology products and process-Matching science and economics. J. appl. Phycol. 4: 267-279.
  • Bourrelly P. 1966/1970. “Les Algues D'eau Douce. Initiation à la systématique”. Tome I: Les Algues vertes. Tome II: Chrysophyées, Xanthophycées et Diatomées. Tome III: Eugléniens, Péridiniens, Algues rouges et Algues bleues. – Avec 118 + 115 + 137 planches, 13+ 22+ 14 figures, 512 + 440 +544pp. Paris: Editions N. Boubée & Cie. doi.org/10.1002/iroh.19740590219
  • Boutte C, Grubisic S, Balthasart P and Wilmotte A. 2006. Testing of primers for the study of cyanobacterial molecular diversity by DGGE. Journal of Microbiological Methods. 65:542-550. doi: 10.1016/j.mimet.2005.09.017
  • Bulut, Y. 2009. Chlorella‘da (Clorophyceae) yağ miktarının arttırma olanaklarının araştırılması. [Yüksek Lisans Tezi]. Çukurova Üniversitesi. 62 s.
  • Chen F. 1996. High cell density culture of microalgae in heterotrophic growth. Trends in Biotechnology. 14:421-426. doi: 10.1016/0167-7799(96)10060-3
  • Chen CY, Yeh KL, Aisyah R, Lee DJ, Chang JS. 2011. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review. Bioresour Technol. 102(1):71-81. doi: 10.1016/j.biortech.2010.06.159
  • Chen CY, Chang JS, Chang HY, Chen TY, Wu JH, Lee WL. 2013. Enhancing microalgal oil/lipid production from Chlorella sorokiniana CY1 using deep-sea water supplemented cultivation medium. Biochemical Engineering Journal. 77:74-81. doi:10.1016/j.bej.2013.05.009
  • Chojnacka K, Marquez-Rocha FJ. 2004. Kinetic and stoichiometric relationships of the energy and carbon metabolism in the culture of microalgae. Biotechnology. 3:21–34. doi:10.3923/biotech.2004.21.34
  • Droop MR. 1974. Heterotrophy of carbon. In: Stewart WDP, editor. Algal Physiology and Biochemistry. Oxford (England). Blackwell Scientific Publ. p. 530-559.
  • Elcik H, Çakmakcı M. 2017. Mikroalg üretimi ve mikroalglerden biyoyakıt eldesi. Journal of the Faculty of Engineering and Architecture of Gazi University. 32(3):795-820. doi: 10.17341/gazimmfd.337627
  • Güner H ve Aysel V. 1991. Tohumsuz Bitkiler Sistematiği, Ege Üniversitesi Fen Fakültesi Kitaplar Serisi No:108
  • Hariskos I, Posten C. 2014. Biorefinery of microalgae opportunities and constraints for different production scenarios. Biotechnol J. 9(6):739-752. doi: 10.1002/biot.201300142
  • Kim BH, Ramanan R, Kang Z, Cho DH, Oh HM, Kim HS. 2016. Chlorella sorokiniana HS1, a novel freshwater green algal strain, grows and hyperaccumulates lipid droplets in seawater salinity. Biomass and Bioenergy. 85:300-305. doi:10.1016/j.biombioe.2015.12.026
  • Kim S, Park JE, Cho YB, Hwang SJ. 2013. Growth rate, organic carbon and nutrient removal rates of Chlorella sorokiniana in autotrophic, heterotrophic and mixotrophic conditions. Bioresour Technol. 144:8-13. doi: 10.1016/j.biortech.2013.06.068
  • Kobayashi M, Kakizono T, Yamaguchi K, Nishio N, Nagai S. 1992. Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions. J Ferment Bioeng. 74 (1):17-20. doi: 10.1016/0922-338X(92)90261-R
  • Lee YK. 2001. Microalgal mass culture systems and methods: their limitation and potantial. Journal of Applied Phyycology. 13:307-315. doi: 10.1023/A:1017560006941
  • Liu J, Huang J, Sun Z, Zhong Y, Jiang Y, Chen F. 2011. Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: assessment of algal oils for biodiesel production. Bioresource Technology. 102:106–110. doi: 10.1016/j.biortech.2010.06.017
  • McLachlan J. 1973. Growth media marine, In Stein, J.R (ed.) Culture Methods&Growth Measurements. Cambridge University Press. p. 25-51.
  • Marquez FJ, Sasaki K, Kakizono T, Nishio N, Nagai S. 1993. Growth characteristics of Spirulina platensis in mixotrophic and heterotrophic conditions. Journal of Fermentation and Bioengineering. 76:408–410. doi: 10.1016/0922-338X(93)90034-6
  • Miao X, Wu Q. 2004. High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides. J Biotechnol. 110(1):85-93. doi: 10.1016/j.jbiotec.2004.01.013
  • Mitra D, van Leeuwen J, Lamsal B. 2012. Heterotrophic/mixotrophic cultivation of oleaginous Chlorella vulgaris on industrial co-products. Algal Res. 1(1):40-48. doi: 10.1016/j.algal.2012.03.002
  • Nübel U, Garcia- Pichel F and Muyzer G. 1997. PCR Primers to amplify 16S rDNA Genes from cyanobacteria. Applied and Environmental Microbiology. 63(8):3327-3332. doi: 10.1128/AEM.63.8.3327-3332.1997
  • Pragya N, Pandey KK, Sahoo PK. 2013. A review on harvesting, oil extraction and biofuels production technologies from microalgae. Renewable Sustainable Energy Rev. 24:159-171. doi: 10.1016/j.rser.2013.03.034
  • Provasoli L. 1963. Growing marine seaweeds. In: De Virville AD and Feldmann J (Eds.) Proceedings of the Fourth International Seaweed Symposium. Pergamon Press. Oxford. p. 9-17.
  • Provasoli L. 1968. Media and prospects for the cultivation of marine algae. In: Watanabe H, Hattori A, (eds.). Cultures and collections of algae. Proceedings US-Japan Conference. Hakone: Japanese Society of Plant Physiology. p. 63-75.
  • Sasson A. 1997. Microalgal biotechnologies: Recent developments and prospects for developing countries. Biotec Publication. 1:76.
  • Sukatar A. 2002. Algal culture methods (in Turkish). Ege Üni Fen Fak. Kitapları Serisi No:184. s.104.
  • Yeh KL, Chang JS, Chen WM. 2010. Effect of light supply and carbon source on cell growth and cellular composition of a newly isolated microalga Chlorella vulgaris ESP-31.Eng Life Sci. 10(3):201-208. doi: 10.1002/elsc.200900116
  • Yıldırım A, Demirel Z, İşleten-Hoşoğlu M, Akgün İH, Hatipoğlu-Uslu S, Conk-Dalay M. 2014. Carotenoid and fatty acid compositions of an indigenous Ettlia texensis isolate (Chlorophyceae) under phototrophic and mixotrophic conditions. Applied Biochemistry and Biotechnology. 172(3): 1307-1319. doi: 10.1007/s12010-013-0599-y
  • Zhang H, Wang W, Li Y, Yang W, Shen G. 2011. Mixotrophic cultivation of Botryococcus braunii. Biomass and Bioenergy 35:1710–1715. doi: 10.1016/j.biombioe.2011.01.002
  • Zhu L. 2015. Biorefinery as a promising approach to promote microalgae industry: An innovative framework. Renewable Sustainable Energy Rev. 41:1376-1384. doi: 10.1016/j.rser.2014.09.040
  • Wang H, Xiong H, Hui Z, Zeng X. 2012. Mixotrophic cultivation of Chlorella pyrenoidosa with diluted primary piggery wastewater to produce lipids. Bioresour Technol. 104:215-220.

Isolation, Molecular Identification, Phototrophic, Mixotrophic and Heterotrophic Production of Chlorella sorokiniana

Yıl 2021, Cilt: 7 Sayı: 2, 128 - 137, 26.08.2021

Döndü Yalçın Bingül , Zeliha Demirel , Meltem Conk Dalay

https://doi.org/10.17216/limnofish.703234

Öz

Microalgae was isolated using dilution and pouring plate method from the sea water sample taken from the Gumuldur region of Izmir. The species was identified as Chlorella sp. through morphologic evaluation under light microscope. Microalgae DNA was isolated through molecular methods and 16S and 18S rRNA gene regions were amplified by means of PCR. As a result of sequencing and phylogenetic evaluation of the gene regions, it was determined that the microalgae was Chlorella sorokiniana. Different methods such as centrifuge washing, antibiotic treatment, growth on agar and single cell isolation used to obtain axenic C. sorokiniana yielded successful results. Biomass and specific growth rate of phototrophic C. sorokiniana (0.19 g L-1 and 0.78 days-1, respectively), mixotrophic C. sorokiniana (0.31 g L-1 and 1.3 days-1, respectively) and heterotrophic C. sorokiniana (0.6 g L-1 and 2.52 days-1, respectively) were determined. According to the findings obtained, the biomass productivity of axenic
C. sorokiniana under different culture conditions can be ordered as: heterotrophy > mixotrophy > phototrophy.

Anahtar Kelimeler

Chlorella sorokiniana molecular identification phototropy mixotrophy heterotrophy

Kaynakça

  • Abreu AP, Fernandes B, Vicente AA, Teixeira J, Dragone G. 2012. Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source. Bioresour Technol. 118:61-66. doi: 10.1016/j.biortech.2012.05.055
  • AlgaeBase. 2021. [Erişim tarihi: 09 Mar 2021]. Erişim Adresi: https://www.algaebase.org/search/species/detail/?species_id=r7b5b5e3003154f99
  • AlgaePARC. 2021. Wageningen University&Research, [Erişim tarihi: 09 Mar 2021]. Erişim Adresi: http://www.algaeparc.com/news
  • Baytut Ö, Gürkanli CT, Gönülol A, Özkoç I. 2014. Molecular phylogeny of Chlorella-related chlorophytes (Chlorophyta) from Anatolian freshwaters of Turkey. Turkish Journal of Botany. 38(3): 600-607. doi:10.3906/bot-1304-32
  • Becker EW. 1995. Microalgae biotechnology and microbiology. Cambridge University Press. p. 293. doi.org/10.1002/jctb.280470214
  • Becker EW. 2007. Microalgae as a source of protein. Biotechnol Adv. 25(2):207-210. doi: 10.1016/j.biotechadv.2006.11.002
  • BLAST. 2021. [Erişim tarihi: 09 Mar 2021]. Erişim Adresi:https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_395406506
  • Borowitzka MA. 1992. Algal biotechnology products and process-Matching science and economics. J. appl. Phycol. 4: 267-279.
  • Bourrelly P. 1966/1970. “Les Algues D'eau Douce. Initiation à la systématique”. Tome I: Les Algues vertes. Tome II: Chrysophyées, Xanthophycées et Diatomées. Tome III: Eugléniens, Péridiniens, Algues rouges et Algues bleues. – Avec 118 + 115 + 137 planches, 13+ 22+ 14 figures, 512 + 440 +544pp. Paris: Editions N. Boubée & Cie. doi.org/10.1002/iroh.19740590219
  • Boutte C, Grubisic S, Balthasart P and Wilmotte A. 2006. Testing of primers for the study of cyanobacterial molecular diversity by DGGE. Journal of Microbiological Methods. 65:542-550. doi: 10.1016/j.mimet.2005.09.017
  • Bulut, Y. 2009. Chlorella‘da (Clorophyceae) yağ miktarının arttırma olanaklarının araştırılması. [Yüksek Lisans Tezi]. Çukurova Üniversitesi. 62 s.
  • Chen F. 1996. High cell density culture of microalgae in heterotrophic growth. Trends in Biotechnology. 14:421-426. doi: 10.1016/0167-7799(96)10060-3
  • Chen CY, Yeh KL, Aisyah R, Lee DJ, Chang JS. 2011. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review. Bioresour Technol. 102(1):71-81. doi: 10.1016/j.biortech.2010.06.159
  • Chen CY, Chang JS, Chang HY, Chen TY, Wu JH, Lee WL. 2013. Enhancing microalgal oil/lipid production from Chlorella sorokiniana CY1 using deep-sea water supplemented cultivation medium. Biochemical Engineering Journal. 77:74-81. doi:10.1016/j.bej.2013.05.009
  • Chojnacka K, Marquez-Rocha FJ. 2004. Kinetic and stoichiometric relationships of the energy and carbon metabolism in the culture of microalgae. Biotechnology. 3:21–34. doi:10.3923/biotech.2004.21.34
  • Droop MR. 1974. Heterotrophy of carbon. In: Stewart WDP, editor. Algal Physiology and Biochemistry. Oxford (England). Blackwell Scientific Publ. p. 530-559.
  • Elcik H, Çakmakcı M. 2017. Mikroalg üretimi ve mikroalglerden biyoyakıt eldesi. Journal of the Faculty of Engineering and Architecture of Gazi University. 32(3):795-820. doi: 10.17341/gazimmfd.337627
  • Güner H ve Aysel V. 1991. Tohumsuz Bitkiler Sistematiği, Ege Üniversitesi Fen Fakültesi Kitaplar Serisi No:108
  • Hariskos I, Posten C. 2014. Biorefinery of microalgae opportunities and constraints for different production scenarios. Biotechnol J. 9(6):739-752. doi: 10.1002/biot.201300142
  • Kim BH, Ramanan R, Kang Z, Cho DH, Oh HM, Kim HS. 2016. Chlorella sorokiniana HS1, a novel freshwater green algal strain, grows and hyperaccumulates lipid droplets in seawater salinity. Biomass and Bioenergy. 85:300-305. doi:10.1016/j.biombioe.2015.12.026
  • Kim S, Park JE, Cho YB, Hwang SJ. 2013. Growth rate, organic carbon and nutrient removal rates of Chlorella sorokiniana in autotrophic, heterotrophic and mixotrophic conditions. Bioresour Technol. 144:8-13. doi: 10.1016/j.biortech.2013.06.068
  • Kobayashi M, Kakizono T, Yamaguchi K, Nishio N, Nagai S. 1992. Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions. J Ferment Bioeng. 74 (1):17-20. doi: 10.1016/0922-338X(92)90261-R
  • Lee YK. 2001. Microalgal mass culture systems and methods: their limitation and potantial. Journal of Applied Phyycology. 13:307-315. doi: 10.1023/A:1017560006941
  • Liu J, Huang J, Sun Z, Zhong Y, Jiang Y, Chen F. 2011. Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: assessment of algal oils for biodiesel production. Bioresource Technology. 102:106–110. doi: 10.1016/j.biortech.2010.06.017
  • McLachlan J. 1973. Growth media marine, In Stein, J.R (ed.) Culture Methods&Growth Measurements. Cambridge University Press. p. 25-51.
  • Marquez FJ, Sasaki K, Kakizono T, Nishio N, Nagai S. 1993. Growth characteristics of Spirulina platensis in mixotrophic and heterotrophic conditions. Journal of Fermentation and Bioengineering. 76:408–410. doi: 10.1016/0922-338X(93)90034-6
  • Miao X, Wu Q. 2004. High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides. J Biotechnol. 110(1):85-93. doi: 10.1016/j.jbiotec.2004.01.013
  • Mitra D, van Leeuwen J, Lamsal B. 2012. Heterotrophic/mixotrophic cultivation of oleaginous Chlorella vulgaris on industrial co-products. Algal Res. 1(1):40-48. doi: 10.1016/j.algal.2012.03.002
  • Nübel U, Garcia- Pichel F and Muyzer G. 1997. PCR Primers to amplify 16S rDNA Genes from cyanobacteria. Applied and Environmental Microbiology. 63(8):3327-3332. doi: 10.1128/AEM.63.8.3327-3332.1997
  • Pragya N, Pandey KK, Sahoo PK. 2013. A review on harvesting, oil extraction and biofuels production technologies from microalgae. Renewable Sustainable Energy Rev. 24:159-171. doi: 10.1016/j.rser.2013.03.034
  • Provasoli L. 1963. Growing marine seaweeds. In: De Virville AD and Feldmann J (Eds.) Proceedings of the Fourth International Seaweed Symposium. Pergamon Press. Oxford. p. 9-17.
  • Provasoli L. 1968. Media and prospects for the cultivation of marine algae. In: Watanabe H, Hattori A, (eds.). Cultures and collections of algae. Proceedings US-Japan Conference. Hakone: Japanese Society of Plant Physiology. p. 63-75.
  • Sasson A. 1997. Microalgal biotechnologies: Recent developments and prospects for developing countries. Biotec Publication. 1:76.
  • Sukatar A. 2002. Algal culture methods (in Turkish). Ege Üni Fen Fak. Kitapları Serisi No:184. s.104.
  • Yeh KL, Chang JS, Chen WM. 2010. Effect of light supply and carbon source on cell growth and cellular composition of a newly isolated microalga Chlorella vulgaris ESP-31.Eng Life Sci. 10(3):201-208. doi: 10.1002/elsc.200900116
  • Yıldırım A, Demirel Z, İşleten-Hoşoğlu M, Akgün İH, Hatipoğlu-Uslu S, Conk-Dalay M. 2014. Carotenoid and fatty acid compositions of an indigenous Ettlia texensis isolate (Chlorophyceae) under phototrophic and mixotrophic conditions. Applied Biochemistry and Biotechnology. 172(3): 1307-1319. doi: 10.1007/s12010-013-0599-y
  • Zhang H, Wang W, Li Y, Yang W, Shen G. 2011. Mixotrophic cultivation of Botryococcus braunii. Biomass and Bioenergy 35:1710–1715. doi: 10.1016/j.biombioe.2011.01.002
  • Zhu L. 2015. Biorefinery as a promising approach to promote microalgae industry: An innovative framework. Renewable Sustainable Energy Rev. 41:1376-1384. doi: 10.1016/j.rser.2014.09.040
  • Wang H, Xiong H, Hui Z, Zeng X. 2012. Mixotrophic cultivation of Chlorella pyrenoidosa with diluted primary piggery wastewater to produce lipids. Bioresour Technol. 104:215-220.
Toplam 39 adet kaynakça vardır.

Ayrıntılar

Birincil Dil Türkçe
Konular Limnoloji
Bölüm Araştırma Makalesi
Yazarlar

Döndü Yalçın Bingül 0000-0003-3255-3753

Zeliha Demirel 0000-0003-3675-7315

Meltem Conk Dalay 0000-0002-1718-7292

Yayımlanma Tarihi 26 Ağustos 2021
Yayımlandığı Sayı Yıl 2021Cilt: 7 Sayı: 2

Kaynak Göster

APA Yalçın Bingül, D., Demirel, Z., & Conk Dalay, M. (2021). Chlorella sorokiniana’ nın İzolasyonu, Moleküler Tanılanması, Fototrofik, Miksotrofik ve Heterotrofik Üretimi. Journal of Limnology and Freshwater Fisheries Research, 7(2), 128-137. https://doi.org/10.17216/limnofish.703234