Abstract
In industrial-scale microalgal cultures, non-target microalgae compete with the desired species for nutrients and CO2, thus reducing the growth rate of the target species and the quality of the produced biomass. Microalgae identification is generally considered a complicated issue; although, in the last few years, new molecular methods have helped to rectify this problem. Among the different techniques available, DNA barcoding has proven very useful in providing rapid, accurate, and automatable species identification; in this work, it is used to assess the genomic identity of the microalga species Scenedesmus sp. ‘almeriensis’, a common strain in industrial-scale cultures. Barcode markers rbcL and ITS1-5.8S-ITS2 were sequenced and the obtained genomic information was used to design a quantitative PCR assay to precisely quantify the S. almeriensis concentration in microalgal cultures of industrial interest. TaqMan chemistry was used to quantify down to 1 μg/L dry weight of S. almeriensis cells, including in the presence of concentrated mixed cultures of other microalgae. A simple direct qPCR approach was also investigated to avoid classic DNA extraction and to reduce total assay time to approximately 2 h. The objective was to design strain-specific tools able to confirm and quantify the presence of different strains in whatever microalgae culture so as to achieve maximal productivity and quality of the produced biomass.
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References
Arnon DI, McSwain BD, Tsujimoto HY, Wada K (1974) Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin. BBA-Bioenerg 357:231–245. https://doi.org/10.1016/0005-2728(74)90063-2
Cao M, Fu Y, Guo Y, Pan J (2009) Chlamydomonas (Chlorophyceae) colony PCR. Protoplasma 235:107–110. https://doi.org/10.1007/s00709-009-0036-9
Cardozo KHM, Guaratini T, Barros MP, Falcão VR, Tonon AP, Lopes NP, Campos S, Torres MA, Souza AO, Colepicolo P, Pinto E (2007) Metabolites from algae with economical impact. Comp Biochem Physiol C Toxicol Pharmacol 146:60–78. https://doi.org/10.1016/j.cbpc.2006.05.007
Carreres BM, de Jaeger L, Springer J, Barbosa MJ, Breuer G, van den End EJ, Kleinegris DMM, Schaffers I, Wolbert EJH, Zhang H, Lamers PP, Draaisma RB, dos Santos VAPM, Wijffels RH, Eggink G, Schaap PJ, Martens DE (2017) Draft genome sequence of the oleaginous green alga Tetradesmus obliquus UTEX 393. Am Soc Microbiol 5:1–2
Coyne KJ, Handy SM, Demir E, Whereat EB, Hutchins DA, Portune KJ, Doblin MA, Cary SC (2005) Improved quantitative real-time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard. Limnol Oceanogr Methods 3:381–391. https://doi.org/10.4319/lom.2005.3.381
Dawidziuk A, Popiel D, Luboinska M, Grzebyk M, Wisniewski M, Koczyk G (2017) Assessing contamination of microalgal astaxanthin producer Haematococcus cultures with high-resolution melting curve analysis. J Appl Genet 58:277–285. https://doi.org/10.1007/s13353-016-0378-x
Dyhrman ST, Erdner D, La Du J, Galac M, Anderson DM (2006) Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR. Harmful Algae 5:242–250. https://doi.org/10.1016/j.hal.2005.07.005
Ebenezer V, Medlin LK, Ki JS (2012) Molecular detection, quantification, and diversity evaluation of microalgae. Mar Biotechnol 14:129–142. https://doi.org/10.1007/s10126-011-9427-y
Fode-Vaughan KA, Wimpee CF, Remsen CC, Lynne M, Collins P (2001) Detection of bacteria in environmental samples by direct PCR without DNA extraction. Biotechniques 31:598–607
Hadi SIIA, Santana H, Brunale PPM, Gomes TG, Oliveira MD, Matthiensen A, Oliveira MEC, Silva FCP, Brasil BSAF (2016) DNA barcoding green microalgae isolated from neotropical inland waters. PLoS One 11:1–12. https://doi.org/10.1371/journal.pone.0149284
Handy SM, Demir E, Hutchins DA, Portune KJ, Whereat EB, Hare CE, Rose JM, Warner M, Farestad M, Cary SC, Coyne KJ (2008) Using quantitative real-time PCR to study competition and community dynamics among Delaware Inland Bays harmful algae in field and laboratory studies. Harmful Algae 7:599–613. https://doi.org/10.1016/j.hal.2007.12.018
Hayden K, Ivors K, Wilkinson C, Garbelotto M (2006) TaqMan chemistry for Phytophthora ramorum detection and quantification, with a comparison of diagnostic methods. Phytopathology 96:846–854. https://doi.org/10.1094/phyto-96-0846
Hebert PDN, Gregory TR (2005) The promise of DNA barcoding for taxonomy. Syst Biol 54:852–859. https://doi.org/10.1080/10635150500354886
Hyka P, Lickova S, Přibyl P, Melzoch K, Kovar K (2013) Flow cytometry for the development of biotechnological processes with microalgae. Biotechnol Adv 31:2–16. https://doi.org/10.1016/j.biotechadv.2012.04.007
Imam SH, Buchanan MJ, Shin HC, Snell WJ (1985) The Chlamydomonas cell wall: characterization of the wall framework. J Cell Biol 101:1599–1607. https://doi.org/10.1083/jcb.101.4.1599
Kim DY, Vijayan D, Praveenkumar R, Han JI, Lee K, Park JY, Chang WS, Lee JS, Oh YK (2016) Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus. Bioresour Technol 199:300–310. https://doi.org/10.1016/j.biortech.2015.08.107
Liu J, Gerken H, Li Y (2014) Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae. J Appl Phycol 26:1719–1726. https://doi.org/10.1007/s10811-013-0220-3
Mingazzini M, Palumbo MT, Mingazzini M, Palumbo MT (2015) Open mass cultures of marine microalgae for biodiesel production: laboratory approach to study species competition in mixed cultures. Nat Resour 6:174–180. https://doi.org/10.4236/nr.2015.63016
Peniuk GT, Schnurr PJ, Allen DG (2016) Identification and quantification of suspended algae and bacteria populations using flow cytometry: applications for algae biofuel and biochemical growth systems. J Appl Phycol 28:95–104. https://doi.org/10.1007/s10811-015-0569-6
Radha S, Fathima AA (2013) Direct colony PCR for rapid identification of varied microalgae from freshwater environment. J Appl Phycol 25:609-613. https://doi.org/10.1007/s10811-012-9895-0
Roa F, Guerra M (2012) Distribution of 45S rDNA sites in chromosomes of plants: structural and evolutionary implications. BMC Evol Biol 12:225. https://doi.org/10.1186/1471-2148-12-225
Maniatis T, Fritsch E, Sambrook J (1982) Molecular Cloning - A Laboratory Manual, First edit. Cold Spring Harbor Laboratory, New York
Sánchez JF, Fernández-Sevilla JM, Acién FG, Cerón MC, Pérez-Parra J, Molina-Grima E (2008a) Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature. Appl Microbiol Biotechnol 79:719–729. https://doi.org/10.1007/s00253-008-1494-2
Sánchez JF, Fernández JM, Acién FG, Rueda A, Pérez-Parra J, Molina E (2008b) Influence of culture conditions on the productivity and lutein content of the new strain Scenedesmus almeriensis. Process Biochem 43:398–405. https://doi.org/10.1016/j.procbio.2008.01.004
Schrader C, Schielke A, Ellerbroek L, Johne R (2012) PCR inhibitors - occurrence, properties and removal. J Appl Microbiol 113:1014–1026. https://doi.org/10.1111/j.1365-2672.2012.05384.x
Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101:87–96. https://doi.org/10.1263/jbb.101.87
Starkenburg SR, Polle JEW, Hovde B, Daligault HE, Davenport KW, Huang A, Neofotis P, McKie-Krisberg Z (2017) Draft nuclear genome, chloroplast genome, and complete mitochondrial genome for the biofuel/bioproduct feedstock species Scenedesmus obliquus strain DOE0152z. Am Soc Microbiol 5:11–12
Toyoda K, Nagasaki K, Tomaru Y (2010) Application of real-time PCR assay for detection and quantification of bloom-forming diatom Chaetoceros tenuissimus Meunier. Plankon Benthos Res 5:56–61. https://doi.org/10.3800/pbr.5.56
Wan M, Rosenberg JN, Faruq J, Betenbaugh MJ, Xia J (2011) An improved colony PCR procedure for genetic screening of Chlorella and related microalgae. Biotechnol Lett 33:1615–1619. https://doi.org/10.1007/s10529-011-0596-6
Wang H, Zhang W, Chen L, Wang J, Liu T (2013) The contamination and control of biological pollutants in mass cultivation of microalgae. Bioresour Technol 128:745–750. https://doi.org/10.1016/j.biortech.2012.10.158
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications. Academic Press, pp 315–322
Woodman ME, Savage CR, Arnold WK, Stevenson B (2016) Direct PCR of intact bacteria (Colony PCR). Curr Protoc Microbiol 42. https://doi.org/10.1002/cpmc.14
Zamora I, Feldman JL, Marshall WF (2004) PCR-based assay for mating type and diploidy in Chlamydomonas. Biotechniques 37:534–536
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This research received funding from the European Union’s Horizon 2020 Research and Innovation program under Grant Agreement No. 727874 SABANA.
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Beatrice-Lindner, P., Garrido-Cardenas, J.A., Sepulveda, C. et al. A new approach for detection and quantification of microalgae in industrial-scale microalgal cultures. Appl Microbiol Biotechnol 102, 8429–8436 (2018). https://doi.org/10.1007/s00253-018-9268-y
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DOI: https://doi.org/10.1007/s00253-018-9268-y