Skip to main content
SpringerLink
Log in

Biochemical characterization and genetic identity of an oil-rich Acutodesmus obliquus isolate

  • Published:
Journal of Applied Phycology Aims and scope Submit manuscript

Abstract

Microalgae provide one of the more promising strategies for the production of renewable biodiesel and liquid fuels. Coupling biodiesel production and wastewater treatment based on the use of microalgae can be an effective approach for achieving sustainable production. Several pilot programs aimed at lipid production are currently working with Acutodesmus obliquus, and companies offering algal inocula are increasing in number. A. obliquus strain RL01 was isolated from a pretreated leachate, and preliminary analyses indicated a high growth rate, a final biomass concentration of 1.9 g∙L-1 and a lipid content of 47% DW. With the aim of strictly differentiating and unequivocally identifying the new strain in comparison with other A. obliquus lipid producers available in culture collections, we employed a DNA barcoding approach, targeting conventional and non-conventional genome regions. Genomic analyses were performed using two internal transcribed spacers (nuclear ITS1 and ITS2 sequences) and three selected chloroplast regions (the trnL genic intron and the rpl20-psbA and psbM-psbZ intergenic spacers). Our findings raise some concerns about the discriminant power of the ITS2 nuclear target and place particular emphasis on the psbM-psbZ plastid DNA barcode, which is characterized by high polymorphism, for use in studies addressing intraspecific genetic diversity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Akase SI, Yoshikawa T, Hayakawa N, Maeda H, Sakata T (2004) Molecular identification of red tide-causing microalga Heterosigma akashiwo strains based on their chloroplast DNA sequences. Fish Sci 70:1043–1050

    Article  CAS  Google Scholar 

  • Buchheim MA, Keller A, Koetschan C, Förster F, Merget B, Wolf M (2011) Internal Transcribed Spacer 2 (nu ITS2 rRNA) Sequence-Structure Phylogenetics: Towards an automated reconstruction of the green algal tree of life. PLoS ONE 6:e16931

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Byun Y, Han K (2006) PseudoViewer: web application and web service for visualizing RNA pseudoknots and secondary structures. Nucleic Acids Res 34:416–422

    Article  Google Scholar 

  • Chase MW, Salamin N, Wilkinson M, Dunwell JM, Kesanakurthi RP, Haidar N, Savolainen V (2005) Land plants and DNA barcodes: short-term and long-term goals. Phil Trans R Soc B 360:1889–1895

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X (2010) Validation of the ITS2 region as a novel DNA Barcode for identifying medicinal plant species. PLoS ONE 5:e8613

    Article  PubMed Central  PubMed  Google Scholar 

  • Chinnasamy S, Bhatnagar A, Hunt RW, Das KC (2010) Microalgae cultivation in a wastewater dominated by carpet mill effluents for biofuel applications. Bioresour Technol 101:3097–3105

    Article  CAS  PubMed  Google Scholar 

  • Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306

    Article  CAS  PubMed  Google Scholar 

  • Clarens AF, Resurreccion EP, White MA, Colosi LM (2010) Environmental life cycle comparison of algae to other bioenergy feedstocks. Environ Sci Technol 44:1813–1819

    Article  CAS  PubMed  Google Scholar 

  • Clarkston BE, Saunders GW (2010) A comparison of two DNA barcode markers for species discrimination in the red algal family Kallymeniaceae (Gigartinales, Florideophyceae), with a description of Euthora timburtonii sp. nov. Botany 88:119–131

    Article  CAS  Google Scholar 

  • Courchesne NMD, Parisien A, Wang B, Lan CQ (2009) Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J Biotechnol 141:31–41

    Article  CAS  PubMed  Google Scholar 

  • da Silva TL, Reis A, Medeiros R, Oliveira AC, Gouveia L (2009) Oil production towards biofuel from autotrophic microalgae semicontinuous cultivations monitorized by flow cytometry. Appl Biochem Biotechnol 159:568–578

    Article  PubMed  Google Scholar 

  • de Cambiaire JC, Otis C, Lemieux C, Turmel M (2006) The complete chloroplast genome sequence of the chlorophycean green alga Scenedesmus obliquus reveals a compact gene organization and a biased distribution of genes on the two DNA strands. BMC Evol Biol. 25:6–37

    Google Scholar 

  • de Morais MG, Costa JAV (2007) Biofixation of carbon dioxide by Spirulina sp. and Scenedesmus obliquus cultivated in a three-stage serial tubular photobioreactor. J Biotechnol 129:439–445

    Article  PubMed  Google Scholar 

  • DeSalle R, Egan MG, Siddall M (2005) The unholy trinity: taxonomy, species delimitation and DNA barcoding. Phil Trans R Soc B 360:1905–1916

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Doria E, Longoni P, Scibilia L, Iazzi N, Cella R, Nielsen E (2012) Isolation and characterization of a Scenedesmus acutus strain to be used for bioremediation of urban wastewater. J Appl Phycol 24:375–383

    Article  CAS  Google Scholar 

  • Doyle JJ (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Evans KM, Wortley AH, Mann DG (2007) An assessment of potential diatom “barcode” genes (cox1, rbcL, 18S and ITS rDNA) and their effectiveness in determining relationships in Sellaphora (Bacillariophyta). Protist 158:349–364

    Article  CAS  PubMed  Google Scholar 

  • Fawley MW, Fawley KP, Hegewald E (2011) Taxonomy of Desmodesmus serratus (Chlorophyceae, Chlorophyta) and related taxa on the basis of morphological and DNA sequence data. Phycologia 50:23–56

    Article  CAS  Google Scholar 

  • Friedrich J, Dandekar T, Wolf M, Müller T (2005) ProfDistS: a tool for the construction of large phylogenetic trees based on profile distances. Bioinformatics 21:2108–2109

    Article  CAS  PubMed  Google Scholar 

  • Griffiths MJ, Harrison STL (2009) Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J Appl Phycol 21:493–507

    Article  CAS  Google Scholar 

  • Grima EM, Medina AR, Giménez AG, Pérez JAS, Camacho FG, Sánchez JLG (1994) Comparison between extraction of lipids and fatty acids from microalgal biomass. J Am Oil Chem Soc 71:955–959

    Article  Google Scholar 

  • Hall JD, Fucikova K, Lo C, Lewis LA, Karol KG (2010) An assessment of proposed DNA barcodes in freshwater green algae. Cryptogam Algol 31:529–555

    Google Scholar 

  • Han J, Zhu Y, Chen X, Liao B, Yao H, Song J, Chen S, Meng F (2013) The short ITS2 sequence serves as an efficient taxonomic sequence tag in comparison with the full-length ITS. BioMed Res Int. doi:10.1155/2013/741476

    Google Scholar 

  • Hegewald E, Wolf M (2003) Phylogenetic relationships of Scenedesmus and Acutodesmus (Chlorophyta, Chlorophyceae) as inferred from 18S rDNA and ITS-2 sequence comparisons. Plant Syst Evol 241:185–191

    Article  Google Scholar 

  • Hegewald E, Bock C, Krienitz L (2013) A phylogenetic study on Scenedesmaceae with the description of a new species of Pectinodesmus and the new genera Verrucodesmus and Chodatodesmus (Chlorophyta, Chlorophyceae). Fottea 13:149–164

    Google Scholar 

  • Hodaifa G, Martínez ME, Sánchez S (2008) Use of industrial wastewater from olive-oil extraction for biomass production of Scenedesmus obliquus. Bioresour Technol 99:1111–1117

    Article  CAS  PubMed  Google Scholar 

  • Johnson JL, Fawley MW, Fawley KP (2007) The diversity of Scenedesmus and Desmodesmus (Chlorophyceae) in Itasca State Park, Minnesota, USA. Phycologia 46:214–229

    Article  Google Scholar 

  • Keller A, Schleicher T, Schultz J, Müller T, Dandekar T, Wolf M (2009) 5.8S-28S rRNA interaction and HMM-based ITS2 annotation. Gene 430:50–57

    Article  CAS  PubMed  Google Scholar 

  • Keller A, Förster F, Müller T, Dandekar T, Schultz J, Wolf M (2010) Including RNA secondary structures improves accuracy and robustness in reconstruction of phylogenetic trees. Biol Direct 5:4

    Article  PubMed Central  PubMed  Google Scholar 

  • Kessler E (1991) Scenedesmus: problems of a highly variable genus of green algae. Bot Acta 104:169–171

    Article  Google Scholar 

  • Khozin-Goldberg I, Cohen Z (2006) The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus. Phytochemistry 67:696–701

    Article  CAS  PubMed  Google Scholar 

  • Kimura M, Crow JF (1964) The number of alleles that can be maintained in a finite population. Genetics 49:725–738

    CAS  PubMed Central  PubMed  Google Scholar 

  • Koetschan C, Hackl T, Müller T, Wolf M, Förster F, Schultz J (2012) ITS2 database IV: interactive taxon sampling for internal transcribed spacer 2 based phylogenies. Mol Phylogenet Evol 63:585–588

    Article  CAS  PubMed  Google Scholar 

  • Kress WJ, Erickson DL (2007) A two-locus global DNA barcode for land plants: the coding rbcL Gene complements the non-coding trnH-psbA Spacer region. PLoS ONE 2:e508

    Article  PubMed Central  PubMed  Google Scholar 

  • Lang I, Hodac L, Friedl T, Feussner I (2011) Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection. BMC Plant Biol 11:124

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Letsch MR, Lewis LA (2012) Chloroplast gene arrangement variation within a closely related group of green algae (Trebouxiophyceae, Chlorophyta). Mol Phyl Evol 64:524–532

    Article  CAS  Google Scholar 

  • Lewontin RC (1972) The apportionment of human diversity. Evol Biol 6:381–398

    Article  Google Scholar 

  • Li Y, Chen YF, Chen P, Min M, Zhou W, Martinez B, Zhu J, Ruan R (2011) Characterization of a microalga Chlorella sp. well adapted to highly concentrated municipal wastewater for nutrient removal and biodiesel production. Bioresour Technol 102:5138–5144

    Article  CAS  PubMed  Google Scholar 

  • Mandal S, Mallick N (2009) Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl Microbiol Biotechnol 84:281–291

    Article  CAS  PubMed  Google Scholar 

  • Mandal S, Mallick N (2011) Waste utilization and biodiesel production by the green microalga Scenedesmus obliquus. Appl Environ Microbiol 77:374–377

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Marcano LBC, Carruyo IM, Montiel XM, Morales CB, Soto PM (2009) Effect of cadmium on cellular viability in two species of microalgae (Scenedesmus sp. and Dunaliella viridis). Biol Trace Elem Res 130:86–93

    Article  CAS  PubMed  Google Scholar 

  • McDevit DC, Saunders GW (2010) A DNA barcode examination of the Laminariaceae (Phaeophyceae) in Canada reveals novel biogeographical and evolutionary insights. Phycologia 49:235–248

    Article  CAS  Google Scholar 

  • Miranda JR, Passarinho PC, Gouveia L (2012) Bioethanol production from Scenedesmus obliquus sugars: the influence of photobioreactors and culture conditions on biomass production. Appl Microbiol Biotechnol 96:555–564

    Article  CAS  PubMed  Google Scholar 

  • Moniz MBJ, Kaczmarska I (2010) Barcoding of diatoms: nuclear encoded ITS revisited. Protist 161:7–34

    Article  CAS  PubMed  Google Scholar 

  • Morsi H, Corradi M, Gorbi G (1998) Toxicity of copper and zinc to two strains of Scenedesmus acutus having different sensitivity to chromium. Environ Exp Bot 40:59–66

    Article  Google Scholar 

  • Müller T, Philippi N, Dandekar T, Schultz J, Wolf M (2007) Distinguishing species. RNA 13:1469–1472

    Article  PubMed Central  PubMed  Google Scholar 

  • Nei M (1973) Analysis of gene diversity in subdivided populations. PNAS 70:3321–3323

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New York, NY

    Google Scholar 

  • Nicolè S, Erickson DL, Ambrosi D, Bellucci E, Lucchin M, Papa R, Kress WJ, Barcaccia G, Donini P (2011) Biodiversity studies in Phaseolus species by DNA barcoding. Genome 54:529–545

    Article  PubMed  Google Scholar 

  • Pittman JK, Dean AP, Osundeko O (2011) The potential of sustainable algal biofuel production using wastewater resources. Bioresour Technol 102:17–25

    Article  CAS  PubMed  Google Scholar 

  • Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818

    Article  CAS  PubMed  Google Scholar 

  • Rai LC, Rai PK, Mallick N (1996) Regulation of heavy metal toxicity in acid-tolerant Chlorella: Physiological and biochemical approaches. Environ Exp Bot 36:99–109

    Article  CAS  Google Scholar 

  • Reuter JS, Mathews DH (2010) RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioin 11:129

    Article  Google Scholar 

  • Richardson B, Orcutt DM, Schwertner HA, Martinez CL, Wickline HE (1969) Effects of nitrogen limitation on the growth and composition of unicellular algae in continuous culture. Appl Microbiol 18:245–250

    CAS  PubMed Central  PubMed  Google Scholar 

  • Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61

    Article  Google Scholar 

  • Rodolfi L, Chini Zittelli G, Bassi N, Padovani G, Biondi N, Bonini G, Tredici MR (2009) Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol Bioeng 102:100–112

    Article  CAS  PubMed  Google Scholar 

  • Ronquist F, Huelsenbeck J and Teslenko M (2011) Draft MrBayes version 3.2 manual: Tutorials and model summaries. http://mrbayes.sourceforge.net/mb3.2_manual.pdf

  • Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496–2497

    Article  CAS  PubMed  Google Scholar 

  • Schultz J, Maisel S, Gerlach D, Müller T, Wolf M (2005) A common core of secondary structure of the internal transcribed spacer 2 (ITS2) throughout the Eukaryota. RNA 11:361–364

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Seibel PN, Müller T, Dandekar T, Wolf M (2008) Synchronous visual analysis and editing of RNA sequence and secondary structure alignments using 4SALE. BMC Res Notes 1:91

    Article  PubMed Central  PubMed  Google Scholar 

  • Selig C, Wolf M, Müller T, Dandekar T, Schultz J (2008) The ITS2 Database II: homology modelling RNA structure for molecular systematics. Nucleic Acids Res 36:D377–380

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sforza E, Cipriani R, Morosinotto T, Bertucco A, Giacometti GM (2012a) Excess CO2 supply inhibits mixotrophic growth of Chlorella protothecoides and Nannochloropsis salina. Bioresour Technol 104:523–529

    Article  CAS  PubMed  Google Scholar 

  • Sforza E, Bertucco A, Morosinotto T, Giacometti GM (2012b) Photobioreactors for microalgal growth and oil production with Nannochloropsis salina: From lab-scale experiments to large-scale design. Chem Eng Res Des 90:1151–1158

    Article  CAS  Google Scholar 

  • Shaw J, Lickey EB, Beck JT, Farmer SB, Liu W, Miller J, Siripun KC, Winder CT, Schilling EE, Small RL (2005) The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. Am J Bot 92:142–166

    Article  CAS  PubMed  Google Scholar 

  • Simionato D, Block MA, Rocca NL, Jouhet J, Maréchal E, Finazzi G, Morosinotto T (2013) Response of Nannochloropsis gaditana to nitrogen starvation includes a de novo biosynthesis of triacylglycerols, a decrease of chloroplast galactolipids and a reorganization of the photosynthetic apparatus. Eukaryot Cell 12:665–676

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C, Valentini A, Vermat T, Corthier G, Brochmann C, Willerslev E (2007) Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding. Nucleic Acids Res 35:e14–e14

    Article  PubMed Central  PubMed  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Torelli A, Marieschi M, Castagnoli B, Zanni C, Gorbi G, Corradi MG (2008) Identification of S2-T A63: A cDNA fragment corresponding to a gene differentially expressed in a Cr-tolerant strain of the unicellular green alga Scenedesmus acutus. Aquat Toxicol 86:495–507

    Article  CAS  PubMed  Google Scholar 

  • van Hannen E, Fink Godhe P, Lurling M (2002) A revised secondary structure model for the internal transcribed spacer 2 of the green algae Scenedesmus and Desmodesmus and its implication for the phylogeny of these algae. Eur J Phycol 37:203–208

    Article  Google Scholar 

  • Wang B, Lan CQ (2011) Biomass production and nitrogen and phosphorus removal by the green alga Neochloris oleoabundans in simulated wastewater and secondary municipal wastewater effluent. Bioresour Technol 102:5639–5644

    Article  CAS  PubMed  Google Scholar 

  • Watterson GA (1975) On the number of segregating sites in genetical models without recombination. Theor Popul Biol 7:256–276

    Article  CAS  PubMed  Google Scholar 

  • Woertz I, Feffer A, Lundquist T, Nelson Y (2009) Algae grown on dairy and municipal wastewater for simultaneous nutrient removal and lipid production for biofuel feedstock. J Environ Eng 135:1115–1122

    Article  CAS  Google Scholar 

  • Wolf M, Achtziger M, Schultz J, Dandekar T, Müller T (2005) Homology modeling revealed more than 20,000 rRNA internal transcribed spacer 2 (ITS2) secondary structures. RNA 11:1616–1623

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wolf M, Ruderisch B, Dandekar T, Schultz J, Müller T (2008) ProfDistS: (profile-) distance based phylogeny on sequence–structure alignments. Bioinformatics 24:2401–2402

    Article  CAS  PubMed  Google Scholar 

  • Xin L, Hong-ying H, Jia Y (2010) Lipid accumulation and nutrient removal properties of a newly isolated freshwater microalga, Scenedesmus sp. LX1, growing in secondary effluent. New Biotechnol 27:59–63

    Article  Google Scholar 

  • Yao H, Song J, Liu C, Luo K, Han J, Li Y, Pang X, Xu H, Zhu Y, Xiao P (2010) Use of ITS2 region as the universal DNA barcode for plants and animals. PLoS ONE 5:e13102

    Article  PubMed Central  PubMed  Google Scholar 

  • Yeh FC, Yang RC, Boyle TBJ, Ye ZH, Mao JX (1997) POPGENE, the user-friendly shareware for population genetic analysis. Mol Biol and Biotechnol Centre Unniv Alberta, Canada, 10

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. TeSAF Department, Plant Pathology Section, University of Padova, Campus of Agripolis - Viale Università 16, 35020, Legnaro, Padova, Italy

    Davide Ferrigo

  2. Laboratory of Genetics and Genomics, DAFNAE, University of Padova, Campus of Agripolis - Viale Università 16, 35020, Legnaro, Padova, Italy

    Giulio Galla & Gianni Barcaccia

  3. Department of Chemical Engineering Principles and Practice “I. Sorgato”, University of Padova, Via Marzolo 9, 35131, Padova, Italy

    Eleonora Sforza

  4. Department of Biology, University of Padova, Via U. Bassi 58/ B, 35121, Padova, Italy

    Tomas Morosinotto & Cristina Ceschi Berrini

Authors
  1. Davide Ferrigo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Giulio Galla
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eleonora Sforza
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tomas Morosinotto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gianni Barcaccia
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Cristina Ceschi Berrini
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cristina Ceschi Berrini.

Additional information

Davide Ferrigo and Giulio Galla authors contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(XLSX 40 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ferrigo, D., Galla, G., Sforza, E. et al. Biochemical characterization and genetic identity of an oil-rich Acutodesmus obliquus isolate. J Appl Phycol 27, 149–161 (2015). https://doi.org/10.1007/s10811-014-0315-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10811-014-0315-5

Keywords

Search

Navigation