High efficiency transformation by electroporation of the freshwater alga Nannochloropsis limnetica

  • Yiwen Chen
  • Hanhua HuEmail author
Original Paper


The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 μg mL−1 of zeocin or 5 μg mL−1 of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10−8 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10–11 × 10−6 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well.

Graphic abstract

High-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times.


Biofuels Electroporation Freshwater microalgae Nannochloropsis limnetica Nuclear transformation 



This work was supported by the National Key R&D Program of China (Grant No. 2018YFD0901500) and National Natural Science Foundation of China (Grant No. 91751117).

Author contributions

HH, taking responsibility for the integrity of the work as a whole, designed the research, analyzed the data and wrote the manuscript. YC performed the experiments and analyze the data. All authors agree on the authorship and submission of the manuscript for peer review.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Informed consent

No informed consent, human or animal rights applicable.

Supplementary material

11274_2019_2695_MOESM1_ESM.doc (37 kb)
Supplementary file1 (DOC 37 kb)


  1. Andersen RA, Brett RW, Potter D, Sexton JP (1998) Phylogeny of the Eustigmatophyceae based upon 18S rDNA, with emphasis on Nannochloropsis. Protist 149:61–74CrossRefGoogle Scholar
  2. Baudelet P-H, Ricochon G, Linder M, Muniglia L (2017) A new insight into cell walls of Chlorophyta. Algal Res 25:333–371CrossRefGoogle Scholar
  3. Chini Zittelli G, Lavista F, Bastianini A, Rodolfi L, Vincenzini M, Tredici MR (1999) Production of eicosapentaenoic acid by Nannochloropsis sp. cultures in outdoor tubular photobioreactors. J Biotechnol 70:299–312CrossRefGoogle Scholar
  4. Falciatore A, Casotti R, Leblanc C, Abrescia C, Bowler C (1999) Transformation of nonselectable reporter genes in marine diatoms. Mar Biotechnol 1:239–251CrossRefGoogle Scholar
  5. Fawley MW, Jameson I, Fawley KP (2015) The phylogeny of the genus Nannochloropsis (Monodopsidaceae, Eustigmatophyceae), with descriptions of N. australis sp. nov. and Microchloropsis gen. nov. Phycologia 54:545–552CrossRefGoogle Scholar
  6. Fietz S, Bleiß W, Hepperle D, Koppitz H, Krienitz L, Nicklisch A (2005) First record of Nannochloropsis limnetica (Eustigmatophyceae) in the autotrophic picoplankton from Lake Baikal. J Phycol 41(4):780–790CrossRefGoogle Scholar
  7. Freire I, Cortina-Burgueño A, Grille P, Arizcun MA, Abellán E, Segura M, Sousa FW, Otero A (2016) Nannochloropsis limnetica: A freshwater microalga for marine aquaculture. Aquaculture 459:124–130CrossRefGoogle Scholar
  8. Gbadamosi OK, Lupatsch I (2018) Effects of dietary Nannochloropsis salina on the nutritional performance and fatty acid profile of Nile tilapia, Oreochromis niloticus. Algal Res 33:48–54CrossRefGoogle Scholar
  9. Jaeger D, Hübner W, Huser T, Mussgnug JH, Kruse O (2017) Nuclear transformation and functional gene expression in the oleaginous microalga Monoraphidium neglectum. J Biotechnol 249:10–15CrossRefGoogle Scholar
  10. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901–3907CrossRefGoogle Scholar
  11. Kilian O, Benemann CSE, Niyogi KK, Vick B (2011) High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proc Natl Acad Sci USA 108(52):21265–21269CrossRefGoogle Scholar
  12. Krienitz L, Wirth M (2006) The high content of polyunsaturated fatty acids in Nannochloropsis limnetica (Eustigmatophyceae) and its implication for food web interactions, freshwater aquaculture and biotechnology. Limnologica 36(3):204–210CrossRefGoogle Scholar
  13. Krienitz L, Hepperle D, Stich H-B, Weiler W (2000) Nannochloropsis limnetica (Eustigmatophyceae), a new species of picoplankton from freshwater. Phycologia 39(3):219–227CrossRefGoogle Scholar
  14. Li F, Gao D, Hu H (2014) High-efficiency nuclear transformation of the oleaginous marine Nannochloropsis species using PCR product. Biosci Biotechnol Biochem 78(5):812–817CrossRefGoogle Scholar
  15. Ma Y, Wang Z, Yu C, Yin Y, Zhou G (2014) Evaluation of the potential of 9 Nannochloropsis strains for biodiesel production. Bioresource Technol 167:503–509CrossRefGoogle Scholar
  16. Noda J, Mühlroth A, Bučinská L, Dean J, Bones AM, Sobotka R (2017) Tools for biotechnological studies of the freshwater alga Nannochloropsis limnetica: antibiotic resistance and protoplast production. J Appl Phycol 29(2):853–863CrossRefGoogle Scholar
  17. Papagianni M, Avramidis N, Filioussis G (2007) High efficiency electrotransformation of Lactococcus lactis spp lactis cells pretreated with lithium acetate and dithiothreitol. BMC Biotechnol 7:15CrossRefGoogle Scholar
  18. Radakovits R, Jinkerson RE, Fuerstenberg SI, Tae H, Settlage RE, Boore JL, Posewitz MC (2012) Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana. Nat Commun 3:686CrossRefGoogle Scholar
  19. Rodolfi L (2009) Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol Bioeng 102(1):100–112CrossRefGoogle Scholar
  20. Siaut M, Heijde M, Mangogna M, Montsant A, Coesel S, Allen A, Manfredonia A, Falciatore A, Bowler C (2007) Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 406(1–2):23–35CrossRefGoogle Scholar
  21. Soria-Verdugo A, Goos E, García-Hernando N, Riedel U (2018) Analyzing the pyrolysis kinetics of several microalgae species by various differential and integral isoconversional kinetic methods and the Distributed Activation Energy Model. Algal Res 32:11–29CrossRefGoogle Scholar
  22. Suchodolskis A, Feiza V, Stirke A, Timonina A, Ramanaviciene A, Ramanavicius A (2011) Elastic properties of chemically modified baker's yeast cells studied by AFM. Surf Interface Anal 43(13):1636–1640CrossRefGoogle Scholar
  23. Sukenik A (1998) Production of eicosapentaenoic acid by the marine eustigmatophyte Nannochloropsis sp. In: Cohen Z (ed) Chemicals from microalgae. Taylor and Francis, London, pp 41–56Google Scholar
  24. Vieler A, Wu G, Tsai CH, Bullard B, Cornish AJ, Harvey C, Reca IB, Thornburg C, Achawanantakun R, Buehl CJ, Campbell MS, Cavalier D, Childs KL, Clark TJ, Deshpande R, Erickson E, Ferguson AA, Handee W, Kong Q, Li X, Liu B, Lundback S, Peng C, Roston RL, Sanjaya Simpson JP, TerBush A, Warakanont J, Zäuner S, Farre EM, Hegg EL, Jiang N, Kuo MH, Lu Y, Niyogi KK, Ohlrogge J, Osteryoung KW, Shachar-Hill Y, Sears BB, Sun Y, Takahashi H, Yandell M, Shiu SH, Benning C (2012) Genome, functional gene annotation, and nuclear transformation of the Heterokont oleaginous alga Nannochloropsis oceanica CCMP1779. PLoS Genet 8(11):e1003064CrossRefGoogle Scholar
  25. Wang D, Ning K, Li J, Hu J, Han D, Wang H, Zeng X, Jing X, Zhou Q, Su X, Chang X, Wang A, Wang W, Jia J, Wei L, Xin Y, Qiao Y, Huang R, Chen J, Han B, Yoon K, Hill RT, Zohar Y, Chen F, Hu Q, Xu J (2014) Nannochloropsis genomes reveal evolution of microalgal oleaginous traits. PLoS Genet 10(1):e1004094CrossRefGoogle Scholar
  26. Wu S, Letchworth GJ (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36(1):152–154CrossRefGoogle Scholar
  27. Zhang C, Hu H (2014) High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation. Mar Genomics 16:63–66CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Key Laboratory of Algal Biology, Institute of HydrobiologyChinese Academy of SciencesWuhanChina
  2. 2.University of Chinese Academy of SciencesBeijingChina

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