WO2018141978A1 - Algal strains - Google Patents
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- WO2018141978A1 WO2018141978A1 PCT/EP2018/052840 EP2018052840W WO2018141978A1 WO 2018141978 A1 WO2018141978 A1 WO 2018141978A1 EP 2018052840 W EP2018052840 W EP 2018052840W WO 2018141978 A1 WO2018141978 A1 WO 2018141978A1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
- C12N1/125—Unicellular algae isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
Definitions
- the present invention relates to algal strains, compositions or taxonomy and in particular but not exclusively to algal compositions or clades for the production of compounds and in particular carotenes, such as ⁇ -carotene.
- Algae are well known, for example there are over twenty strains of algae assigned to the genus, Dunaliella, have been isolated from various hypersaline environments. Dunaliella is one of the richest sources of natural ⁇ -carotene.
- Dunaliella salina is a type of halophile green micro-algae. Halophiles are organisms living and fostering in environments of high salinity. D. salina is known for its high concentration of carotenoids that provide protection against the intense light normally measured in salt evaporation ponds. Due to its carotenoid accumulation, D. salina has various applications in health and nutrition.
- Dunaliella salina is a rich source of natural orange-, yellow- or red-pigmented carotenoids. Their antioxidant and pro-retinoic acid activity may protect humans from compromised immune response, premature ageing, cancers, cardiovascular disease and arthritis.
- Duniella salina consists of a species complex made up of a diverse range of isolates collected from natural and man-made hypersaline environments. Strains are routinely erroneously assigned to and within this complex.
- Dunaliella (Chlorophyceae, Dunaliellales) is a genus of algae with immense economic potential owing to its production of an array of exploitable compounds, including ⁇ -carotene, glycerol and phytosterols. Microalgal species within this genus include halophilic and halotolerant strains, with D. salina able to tolerate NaCI saturation of approximately 5.5 M NaCI. Dunaliella lacks a polysaccharide cell wall and has a flexible cell membrane capable of rapidly changing shape in response to osmotic stress. Cell size is also highly variable with differences in cell size related to growth conditions, e.g. nutrients, pH and salt concentration and can also vary within the same culture at the same stage of growth, for example D. salina exhibits 5-29 ⁇ m cell length, 3.8-20.3 ⁇ m width).
- Dunaliellaceae are four sections of Dunaliella; section Tertiolectae which are oligo- euhaline and do not accumulate carotenes and grow at an optimum salinity of ⁇ 6 NaCI; section Dunaliella which are halophilic species that accumulate carotenes; section Virides which are hyperhaline, always green and radially symmetrical; and Peirceinae which are hyperhaline, always green but cells are bilaterally symmetrical.
- D. salina D. parva and D. pseudosalina as well as D. bardawil, which is also considered as D. salina in some studies.
- D. salina D. parva
- D. pseudosalina as well as D. bardawil
- salina is particularly polymorphic, with cells pigmented green or red, dependent on the amount of ⁇ -carotene accumulated. Under high stress conditions, members of this species can accumulate >5 ⁇ -carotene dry weight. D. bardawil was originally reported to accumulate considerably larger amounts of ⁇ -carotene compared with D. salina, accumulating the ⁇ -carotene in membrane-free globules in the interthylakoid spaces of the chloroplast.
- D. parva typically accumulates up to 5% ⁇ -carotene dry weight and D. pseudosalina cells can be pigmented yellow/orange due to the accumulation of canthoxanthin as their main carotenoid pigment, as opposed to ⁇ -carotene.
- the section Virides is a large group, encompassing I I species. However, four of these species (D. baas- beckingii, D. media, D. ruineniana and D. gracilis) have been described based on a single field collection so are essentially provisional names. Variations in cell shape and size, flagella length and stigma are criteria often used to delineate these species from each other, e.g. D. bioculata has 2 stigmata whereas the other species have I . Members of the Virides are often difficult to classify purely based on morphology, with D. viridis and D. minuta being particularly difficult to resolve due to their similar cell size and the ambiguous characteristic of cell form, defining these two from each other.
- ITS I The hypervariable regions, ITS I , 5.8S rRNA and ITS 2 have been frequently employed by molecular studies of this important algal group; interestingly, some studies have favoured to use each spacer sequence separately in phylogenetic analysis.
- Assun ao et al. 2012 undertook a comprehensive analysis of 3 Dunaliella species using ITS 2, identifying 5 main clades, viridis, salina I, salina II, tertiolecta and pseudosalina, with the latter clade being somewhat ambiguous due to problems with taxonomy.
- ITS markers are often favoured for identification as there are many copies in the genome making it easy to amplify, it is biparentally inherited and insertions/deletions within the sequence are common meaning there is good variability between species.
- Other nuclear markers used for the analysis of Dunaliella from hypersaline environments include the large subunit rDNA and small subunit rDNA and these have shown good potential for taxonomic resolution.
- Both rbcL and tufA are plastid genes, with tufA encoding elongation factor Tu, and has become more frequently used for molecular studies of algae. Availability of sequences, however, is limited with only two sequences for Dunaliella sp. available in Genbank, making phylogenetic analysis less conclusive compared to other markers. Moniz et al., 2014 used the tufA gene in their analysis of the order Prasiolales (Chlorophyta) finding that there was good agreement between the phylogenies of tufA, rbcL and psaB. Presently, tufA as a marker for Dunaliella taxonomy has not been thoroughly examined and its potential not fully realised. rbcL analysis has typically not highlighted intraspecific variation to the same degree as other markers such as the ITS regions.
- Suitable molecular tools are needed for accurate identification of species as this will aid in accurately identifying those isolates that will be economically valuable, e.g. strains of D. salina that produce high levels of 9-cis- - carotene that is economically more valuable than its isomer all-trans- -carotene, and to further understand the molecular evolution of this important group.
- This study set out to genetically characterise a range of Dunaliella isolates collected from a range of geographical provinces including Israel, Spain, South Africa, Italy and Sun. We sought to employ a suite of molecular tools to provide a comprehensive analysis of different markers and their suitability for application to the genus Dunaliella.
- Dunaliella strains from Eilat, Israel and Monzon, Spain were isolated and characterised by the Marine Biological Association, UK (https://www.mba.ac.uk/). These strains are now available by application to the MBA Culture Collection.
- Dunaliella salina strain DF 15 isolated from Eilat has a significantly higher cellular content and higher productivity of carotenoids than many other hyper-accumulating carotenogenic strains such as D. bardawil UTEX 2538.
- Dunaliella salina strain DF40 isolated from Eilat is very similar to D. bardawil and grows in very highly saline water associated with crystallizing salt ponds.
- Some aspects of the present invention relate to halotolerant hyper-accumulating carotenogenic strains of Dunaliella.
- An aspect of the present invention provides a new composition or taxonomy containing algae from the sections Dunaliella and Virides.
- a further aspect provides an algal composition or taxonomy comprising, consisting of or including D. rubeus, D. salina aureus and D. velox.
- a further aspect provides a composition or taxonomy containing algae from the genus Dunaliella in a body of aqueous nutrient solution, in which the algae is from a clade other than the D. bardawil and salina salina clades.
- composition or taxonomy may be provided in a salt solution.
- the salt solution may contain less than 6% NaCI.
- a further aspect provides a method of strain selection or determination for the production of ⁇ - carotene by growing alga of the genus Dunaliella and extracting the ⁇ -carotene produced thereby.
- the alga may be from a clade other than the previously described D. bardawil and D. salina salina clades.
- the alga may be selected from the group comprising: (a) D. salina aureus SA3, SA4, T68, T4 I , T36, and T37 (b) D. rubeus DF I 5 (c) D. velox SA5, SA6.
- An aspect of the present invention provides a method of producing ⁇ -carotene by growing alga of the genus Dunaliella and extracting the ⁇ -carotene produced thereby.
- the alga may be from a clade other than the previously described D. bardawil and D. salina salina clades.
- the alga may be selected from the group comprising: (a) D. salina aureus SA3, SA4, T68, T4 I , T36, and T37 (b) D. rubeus DF 15 (c) D. velox SA5, SA6.
- a further aspect provides an algae bio-refinery, for example a microalgae-based biorefinery, comprising or including one or more strains of algae described herein for the production of one or more (useful) compounds.
- Some aspects of the present invention relate to an algae biorefinery, based on biomass from the halotolerant microalga Dunaliella salina.
- the biorefinery elements may be integrated and optimised using sustainability assessments.
- the biorefinery elements include:
- Biomass New strains cultivated in lakes, raceways and photobioreactors.
- Bioprocessing Key biomass processing technologies are applied to the biomass.
- Some aspects include innovative spiral plate technology for dynamic settling:
- the collected algal biomass After harvesting, the collected algal biomass, usually a liquid suspension, should be processed immediately. If it has to be shipped for processing it should be dried to avoid degradation of biomass and costs of transporting water.
- Two different methods include:
- Freeze-drying or lyophilisation a dehydration process which involves freezing the material and then reducing the surrounding pressure to allow the frozen water to sublimate (from solid to gas).
- the present invention utilizes three harvesting systems for Dunaliella biomass, depending on the end- products required:
- Stacked disk clarifier centrifuges Dunaliella cells are enriched in ⁇ -carotene and lack of glycerol and other water soluble compounds.
- Dunaliella cells are rich in ⁇ -carotene with predominantly intact cells.
- Membrane filtration Dunaliella cells enriched in ⁇ -carotene retain on membrane, while solutes as well as bacteria and viruses pass through.
- aspects and embodiments of the present invention may comprise, consist of or include: (i) process of isolating and characterizing a new microorganism; (ii) new microorganism as produced by a defined process; (iii) new microorganism per se; and (iv) process of cultivation or otherwise using a known or new microorganism to: (a) a form of multiplied microorganism itself, for example biomass, and (b) a by- product of microbial growth, for example a useful industrial product.
- D. viridis (Borowitzka & Siva, 2007); D. peircei UTEX 2192 has been proposed as D. maritima (Borowitzka & Siva, 2007) and D. viridis (Assuncao et al., 2012).
- Figure 3 Neighbour-joining tree of Dunaliella strains isolated during this study and sequences from Genbank based on a 521 bp alignment of the rbcL gene. Bootstrap values were retrieved from 1000 replicates and those >70 are indicated at the nodes for neighbour-joining, maximum likelihood and Bayesian respectively. Strain names followed by an asterisk indicate proposed taxonomic changes, where D.
- D. salina bardawil UTEX 2538 and ATCC 30861 has been proposed as D. salina (Borowitzka & Siva, 2007); D. bioculata UTEX 199 has been proposed as D. tertiolecta (Assuncao et al., 2012); D. salina UTEX 200 has been proposed as D. viridis (Gonzalez et al., 2001 ; Borowitzka & Siva, 2007).
- Figure 4 Neighbour-joining tree of Dunaliella strains isolated during this study and sequences from Genbank based on a 6 l4bp alignment of the tufA gene. Bootstrap values were retrieved from 1000 replicates and those >70 are indicated at the nodes for neighbour-joining, maximum likelihood and Bayesian respectively. Strain names followed by an asterisk indicate proposed taxonomic changes, where D. bardawil UTEX 2538 and ATCC 30861 has been proposed as D. salina (Borowitzka & Siva, 2007).
- Figure 5 Neighbour-joining tree of Dunaliella strains isolated during this study and sequences from Genbank based on an 530bp alignment of the ITS 1 , 5.8S, ITS2. Bootstrap values were retrieved from 1000 replicates and those >70 are indicated at the nodes for neighbour-joining, maximum likelihood and Bayesian respectively. Strain names followed by an asterisk indicate proposed taxonomic changes, where D. bardawil UTEX 2538 and ATCC 30861 has been proposed as D. salina (Borowitzka & Siva, 2007); D. bioculata UTEX 199 has been proposed as D. tertiolecta (Assuncao et al., 2012); D.
- peircei CCAP 19/2 has been proposed as D. tertiolecta (Gonzalez et al., 2001 ); D. parva SAG 19- 1 has been proposed as D. maritima (Borowitzka & Siva, 2007) and D. viridis (Assuncao et al., 2012); D. salina CCAP 19/3 has been proposed as D. viridis (Borowitzka & Siva, 2007); Dunaliella sp. ABRIINW M I/ I has been proposed as D. viridis (Assuncao et al., 2012).
- Figure 6 Neighbour-joining tree of Dunaliella strains isolated during this study and sequences from Genbank based on an alignment of a concatenation of the ITS-LSU-rbcL-tufA sequences used to produce Fig. 2-Fig. 5. Bootstrap values were retrieved from 1000 replicates and those >70 are indicated at the nodes for neighbour-joining, maximum likelihood and Bayesian respectively. Images show the morphology of representative strains and the scale bar is equivalent to 25 ⁇ m. Strain names followed by an asterisk indicate proposed taxonomic changes, where D. bardawil UTEX 2538 and ATCC 30861 has been proposed as D. salina (Borowitzka & Siva, 2007).
- Figure 7 CLUSTALW alignment of the V9 SSU sequences generated in this study and sequences from Genbank. Dots 5 17 indicate identical nucleotides and letters indicate nucleotide substitutions.
- Figure 8 Maximum likelihood phylogenetic tree based on ITS I -ITS2, rbcL, 28S rDNA & I 8S rDNA sequences.
- Figure 9 Growth curves for DF I 5 (red line) and DF I 7 (green line) where arrows indicate timings of RNA extractions for transcriptome sequencing. Inserts: Images of DF 15 (red box) and DF 17 (green box).
- Figure 10 Microscopy observation of Dunaliella cells and photographs of stationary phase cultures of CCAP 19/30, UTEX 2538, DF 17, DF40 and DF 15 grown under a light intensity of 100-200 M mol m -2 s -1 at 20°C.
- DIC Differential interference contrast
- Figure I I Growth curves for the five Dunaliella strains: (a) CCAP 19/30; (b) DF I 5; (c) DF I 7; (d) DF40; (e) UTEX 2538 each grown under four identical light intensities of 200, 500, 1000 and 1500 ⁇ m ⁇ m -2 s 1 at a light/dark cycle of 12 h light and 12 h dark (LD200, LD500, LD 1000 and LD 1500); (f) specific growth rates of each strain grown under the four light intensities. Each culture condition was set up in triplicate.
- Figure 13 Cellular content of total chlorophyll (a) and total carotenoids (b) of the five Dunaliella strains grown under four light intensities of 200, 500, 1000, and 1500 ⁇ m ⁇ m -2 s -'. Samples were taken at the mid log phase and all culture conditions were repeated at least in triplicates.
- Figure 14 HPLC chromatograms of MTBE/ethanol extracts of the five Dunaliella strains cultivated under 1500 ⁇ m ⁇ m ⁇ 2 s " '. The major peaks shown are: ( I ) lutein, (2) zeaxanthin, (3) all-trans ct-carotene, (4) all-trans ⁇ -carotene and (5) 9-c/s ⁇ -carotene.
- Figure I Cellular contents of (a) all-trans ⁇ -carotene, (b) 9-cis ⁇ -carotene, (c) lutein, (d) zeaxanthin, (e) all-trans a-carotene and (f) glycerol in the five Dunaliella strains cultivated under four light intensities of 200, 500, 1000, and 1500 ⁇ m ⁇ m -2 s -1 . Samples were taken at the mid log phase and all culture conditions were repeated at least in triplicate. Figure 16.
- I I traits all-trans ⁇ -carotene, 9-c/s ⁇ -carotene, glycerol, lutein, zeaxanthin, all-trans ⁇ -carotene, photosynthesis, respiration, total carotenoids, total chlorophyll, and specific growth rate
- I I traits all-trans ⁇ -carotene, 9-c/s ⁇ -carotene, glycerol, lutein, zeaxanthin, all-trans ⁇ -carotene, photosynthesis, respiration, total carotenoids, total chlorophyll, and specific growth rate
- Table I Dunaliella sp. isolates collected during this study.
- Table 3 Results from intron sizing and sequence alignments of introns.
- DF48, SA7, T32, T34, T75 and T76 the anterior of the cell was colourless and free of chloroplast with the pyr enoid often clearly defined in the basal cell, furthermore, DF48, SA7, T32, had the presence of 2 stigmata in the anterior part of the cell, a feature described in D. bioculata, a member of the Virides. SA5 and SA6 were slightly larger than the aforementioned green strains (7.61 -8.13 ⁇ m length; 5.49-6.41 ⁇ m width). Cells were oval/pyriform and the stigma could be identified.
- the large-subunit phylogeny distinguished three virides clades supported by high bootstrap values (Fig. 2) with SAI and SA2 clustered with a known species of D. viridis.
- the LSU phylogeny created a large clade of strains assigned as D. salina and D. tertiolecta with weakly supported subclades, owing to only a small number of nucleotide substitutions between strains of D. tertiolecta and D. salina, e.g. D. tertiolecta UTEX 199 has 2bp difference across the 477bp alignment with D. salina DF41.
- SA5 and SA6 were separated and clustered with D.
- salina which follow the clustering patterns described by Assun ao et al. (2012) (salina I and salina II).
- the rbcL phylogeny is in agreement with the LSU phylogeny in that it forms strongly supported clade of SA5 and SA6 and three Virides clades.
- T32 clusters differently within the LSU phylogeny (with T76, T34 and T77), compared to the rbcL phylogeny where it is identical to SA7 and DF48.
- the tufA phylogeny (Fig. 4) strongly supports the clustering of D. tertiolecta in a separate clade to the D. salina clade which also encompasses the strains, SA5 and SA6.
- the Virides form 3 strongly supported subdivisions separated by long branch lengths with the distance between T76 and DF48 equating to approximately 3 1 nucleotide substitutions between the two subclades (comparing 614bp sequence).
- the phylogenetic tree based on ITS I , 5.8S and ITS2 (Fig. 5) provided much more resolution compared to the other trees and was a robust tree supported by high bootstrap values.
- Two salina groups were identified, according to Assun ao et al. (2012) and these were clearly separated by long branch lengths. Due to ambiguous bases in the DF 15 sequences, this amplicon was cloned and sequenced, hence the inclusion in the tree of four DF I 5 sequences.
- DF I 5 clones were found to have nucleotide substitutions across the ITS region when sequenced with the three sequences identified found to share >99% identity. Whilst grouping within the salina clade, DF I 5 does not cluster within either of the sub clades classified as salina I or II by Assun ao et al. (2012). SA5/6 formed a separate clade with tentatively characterised D. viridis, D. maritima and D. tertiolecta. In all four trees, SA I and 2 clustered together forming a clade with D. viridis sequences from genbank (where available) that was separated by significant genetic distance and supported by high bootstrap values.
- ITS-LSU-rbcL-tufA (Fig. 6) was constructed to further resolve/affirm any clades/sub-clades to provide reliable information on the taxonomy of these strains in combination with microscopy. It is important to note that in this tree, only sequences that were generated in this study were used as it was difficult to confidently match sequences from genbank that were derived from the same culture, particularly due to mis- identification and cross contamination problems. Three sub-clades of the section Dunaliella were detected that morphologically were identified as D. salina, and these were supported by significant bootstrap values. Within each of the subclades the strains shared 99% identity, with all the strains morphological identified as D.
- D. tertiolecta strains (section Tertiolectae) shared 99% identity and 97% identity with members of the section Dunaliella, with SA5/6 sharing 97% and 98% identity with members of section Dunaliella and Tertiolectae respectively.
- a well delineated Virides section could be identified supported by high probability, with members of this section sharing 91 -93% identity with members of the Tertiolectae and Dunaliella.
- the three clades identified within the Virides showed greater divergence with SA I/2 (D. viridis) sharing 91 % with the DF48 clade (D. bioculata) and 92% identity with the T75 clade (D. minuta).
- V9 amplicon sequencing was undertaken for DF 15, DF 17, DF40, D. bardawil UTEX 2538, Dunaliella sp.
- V9 primers used were found to not only amplify Dunaliella DNA but also bacterial DNA (cultures were non-axenic) and hence PCR products required cloning prior to sequencing to provide good quality sequences.
- the intron sizing method revealed useful information regarding the history of Dunaliella spp. in culture collections. Olmos et al. (2009) concluded cross contamination of cultures had occurred resulting in differences in amplicon sizes in what was thought to be the same culture. Our study confirms that, based on sequencing analysis of 4 marker genes, the current CCAP 19/30 is in fact a strain of D. tertiolecta, not D. salina. With regards to the supposedly same cultures of D. bardawil, strain UTEX 2538 had I intron and strain ATCC 30861 had 0 introns indicating they are not the same.
- salina have no introns (the same as tertiolecta) negates the intron-sizing as a tool for separating these strains from each other as both an isolate of tertiolecta and salina would produce the same size amplicon despite these two being very different species.
- the V9 region does not offer enough variability for accurate taxonomy for the Dunaliella species.
- the V9 region lends itself to next generation sequencing methodologies, due to its heterogeneity and short length, however, the fact that 3-4 groups cannot be resolved using this marker raises questions on its suitability for analysis of hypersaline microbial communities as a significant portion of Dunaliella diversity will be missed.
- the V2-V4 region of the I 8S was found to have best phylogenetic resolution compared to the other hypervariable regions in dinoflagellates
- the section Dunaliella contains hyperhaline species that accumulate carotenes and includes the species, D. salina, D. pseudosalina and D. parva.
- Two clusters of D. salina have been previously identified, e.g. salina I and salina II based on the ITS2 sequences (Assun ao et al. 2012). Strains isolated during this study fall into both these groups which can be distinguished from each other based on rbcL, LSU and ITS.
- One distinctive D. salina strain DF 15 was significantly different and does not delineate with either groups.
- D. salina a family of the previously identified salina clade II (Assun ao et al. 2012) to be referred to as D. salina.
- This group contains the original isolate of what we know as D. salina, isolated in 1967 by Loezing from Baja California and is deposited within the UTEX culture collection as # 1644. However, this species should be subdivided into D. salina salina and D.
- D. bardawil the first isolate within this clade
- D. bardawil the first isolate within this clade
- the genetic data supports the delineation D. bardawil from D. salina. Whilst some studies have reported similarities between these species (Jahnke et al. 1999; Borowitzka and Siva 2007), other studies using genetically verified strains of D. salina and D.
- D. pseudosalina cells are classified as green to orange in colour with wide cylindrical, oval shaped cells exhibiting radial symmetry. Cells range from I I- 23 ⁇ length; 6- 16 ⁇ m width with flagella 1.5 x cell length and a large distinctive elongated stigma (Massyuk 1973). D. parva can turn orange/red under high stress typically accumulating up to 5% ⁇ carotene dry weight (less than D. salina) and have a distinct stigma (Massyuk 1973).
- SA5 and SA6 are smaller cells that resemble those of the viridis clade rather than D. pseudosalina and furthermore do not accumulate ⁇ carotene as one would expect in D. parva.
- the phylogenetic analysis clearly separates them from the other D. viridis sequences available, assigned as viridis clade I by Assun ao et al. (2013), with significant genetic distance.
- SA5 and 6 have a unique intron further supporting they are a distinct clade.
- T77 is significantly smaller than the other cells in this clade and is more spherical in shape and fits with the description of D. minutissima yet genetically is not distinct. Therefore, we propose that D. minutissima is actually another morphological variation of D. minuta.
- This minuta clade shares 96% identity with the DF48, SA7 and T32 clade but is separated by high bootstrap support (interestingly T32 groups with minuta for rbcL only). Whilst the cell size and shape are similar, in the case of DF48, SA7 and T32, they appear to have 2 eye spots and furthermore the intron classification separates the groups, indicating they are genetically distinct.
- strains deposited in culture collections require updating to reflect new information on their taxonomic affiliation as well as Genbank entries.
- D. bardawil ATCC 30861/UTEX 2538/CCAP 19/30 which is supposedly the same strain, we strongly recommend that when a strain is acquired from a culture collection basic genetic screening (ITS) is warranted to confirm the identity of the strain and thus adding integrity to the scientific study.
- ITS basic genetic screening
- DNA was extracted from 10 mL late exponential cultures using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer's instructions with the exception of the elution volume that was 50 ul.
- PCR was carried out using a suite of primers (Table 2) in a Corbett Thermocycler. PCR reactions were typically carried out in 50 ⁇ volumes containing 2 ⁇ DNA, 25pmol each primer, I x buffer 2.5mM MgCI2, 0.0025mM dNTPs, I Unit Gotaq polymerase (Promega) unless otherwise stated (Table 2).
- PCR reactions proceeded with an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 54°C for 45 seconds and extension at 72°C for I minute unless otherwise stated (Table 2).
- PCR reactions had a final extension step of 72°C for 5 minutes.
- PCR products were either sequenced directly using the respective primers (source bioscience) or in some cases cloning was necessary to ensure a single sequence was obtained. In these instances, PCR products were electrophoresed on a 1.2% (w/v) agarose gel in I x TAE and purified using the Zymoclean gel purification kit (Cambridge Biosciences).
- ⁇ ⁇ purified PCR product was ligated into the pCR2.1 TA cloning vector (Invitrogen) and transformed according to manufacturer's instructions. Sequences were manually verified for quality using Chromas (Technelysium Pty Ltd). Multiple sequence alignments were constructed in BioEdit 7.0 (Hall 1999) using ClustalW with other available sequences from Genbank. Phylogenetic analysis based on neighbour-joining and maximum likelihood was undertaken using MEGA 6 (Tamura et al. 2013) and Bayesian using Geneious (Kearse et al. 2012). Bootstrap values were retrieved from 1000 replicates.
- the raw sequence data obtained from the cDNA libraries were pooled and subjected to filtering and trimming of adaptors for cDNA synthesis, primers, poly (A/T) tails and potential contaminating vector sequences. Following the sequence trimming, the reads for each of the DF strains were assembled together using SeqMan NGen v 14 (DNASTAR). A total 86 million reads survived the QC filtering, assembled into 50,922 transcripts of an average length of 1 ,445kb with 32,383 having a size > I kb.
- Example 3 D. salina UTEX 2538 was obtained from the Culture Collection of Algae at The University of Texas at Austin (UTEX, Austin, TX, USA) and D. salina CCAP 19/30 was obtained from the Culture Collection of Algae and Protozoa at Scottish Marine Institute (CCAP, Scotland, UK). D-Factory strains DF 15 and DF 17 were isolated from a salt pond in Eilat, Israel, and DF40 was isolated from a salt pond in Monzon, Spain.
- the new isolates were identified as strains of or closely related to Dunaliella salina (bardawil) by The Marine Biological Association (MBA, Madison, Devon, UK) and are now deposited at the MBA culture collection (www.mba.ac.uk/culture-collection/). Algae were cultured in Modified Johnsons Medium (Borowitzka 1988) containing 10 mM NaHC0 3 with the pH value adjusted to 7.5 with 10 mM Tris-buffer, and 1.5 M NaCI, which has been tested as the optimal salinity for cell growth of the strains.
- Cultures were maintained in a temperature-controlled growth chamber at 20 ⁇ 2 °C with illumination provided under a 12 h light, 12 h dark cycle ( 12/ 12 LD) by white light emitting diode (LED) lights with a light intensity of ⁇ 200 ⁇ m ⁇ photons m -2 s -1 .
- 12 h light, 12 h dark cycle 12/ 12 LD
- LED white light emitting diode
- the Eclipse Ti-U inverted research microscope (Nikon, Tokyo, Japan) with a Nikon Digital Sight DS-Fi l camera system was used to take brightfield microscopy photographs of cells of each Dunaliella strain.
- the objective lens used was Nikon Splan Fluor ELWD 60x/0.7 and the ocular lens was Nikon CFI I Ox/22.
- the NIS-Elements Advanced Research Microscope Imaging Software (Nikon, Tokyo, Japan) was used to acquire the photos.
- Differential interference contrast (DIC) microscopy photographs were also obtained using a confocal microscope system ZEISS LSM 880 (Carl Zeiss Microscopy GmbH, Jena, Germany).
- the ZEISS Plan Apochromat 63x/ l .4 oil DIC objective lens and the Carl Zeiss PI 10x/23 ocular lens were used. Images were acquired and analyzed through the ZEN 2.1 LSM software (Carl Zeiss Microscopy GmbH).
- compositions of pigments extracted from different strains were analyzed using high performance liquid chromatography (HPLC) with diode array detection (DAD) (Agilent Technologies 1200 series, Agilent, Santa Clara, CA, USA).
- DAD diode array detection
- Carotenoid standards of all-trans a-carotene, all-trans ⁇ -carotene and zeaxanthin were obtained from Sigma-Aldrich, Inc. (Merck KGaA, Darmstadt, Germany). Lutein and 9- cis ⁇ -carotene were obtained from Dynamic Extractions (Tredegar, UK).
- Carotenoids and chlorophylls were extracted from freshly harvested cells using methyl tert-butyl ether (MTBE) and Methanol (MeOH) (20:80) as extraction solvent.
- 15 mL of algal culture was centrifuged at 3000 g at 18 °C for 5 min and the pellet was extracted with 10 mL MTBE-MeOH (20:80) and sonicated for 20 s.
- the sample was clarified by centrifugation at 3000 g at 18 °C for 5 min, then I -2 mL of the supernatant was filtered through 0.45 ⁇ syringe filter into amber HPLC vials.
- Hierarchical cluster analysis is based on the strength of the correlations and the distance in the clustering dendrogram reflects the dissimilarity among these parameters. Traits examined with strong correlations are grouped as a cluster. A principle component analysis was carried out using the whole data set to reveal the relatedness between the examined traits.
- HPLC-DAD was used to quantify the contents of major carotenoids, namely lutein, zeaxanthin, all-trans ⁇ -carotene, 9-cis ⁇ -carotene, and all-trans ct-carotene, in each strain acclimated in response to four light intensities, to understand the effect of light in carotenoid metabolism.
- Figure 14 shows HPLC chromatograms of the pigment extracts from the five Dunaliella strains grown under the light intensity of 1500 ⁇ m ⁇ m ⁇ 2 s ⁇ '. It is clear that CCAP 19/30 does not accumulate ⁇ -carotene even under high light intensity.
- DF I 5, DF40 and UTEX 2538 have a similar pigment profile and ⁇ -carotene dominates the carotenoid composition.
- DF 17 produced a higher relative amount of zeaxanthin under high light stress compared with the other strains, indicating the important role of zeaxanthin in DF 17 for photoprotection.
- UTEX 2538 already known to be a massive carotene- accumulating strain, had faster growth rates than DF 15 under all light intensities examined, as shown in Figure I If.
- DF I 5 accumulated a high carotene content even under the lowest light intensity tested here.
- variation in ⁇ -carotene content has been reported to correlate with the integral irradiance received during a division cycle and to be a specific mechanism of photoprotection, which may explain why DF 15 has a higher cellular content of ⁇ -carotene than UTEX 2538.
- DF 15 has the advantage of accumulating a large amount of ⁇ -carotene even without light stress ( Figure 15), and also highest productivity of both all-trans and 9-cis ⁇ -carotene under light stress, therefore has great potential for the commercial production of ⁇ -carotene with less light energy input required.
- DF 17 had the highest lutein content at 1000 ⁇ m ⁇ m -2 s -1 , and the lowest at 1500 ⁇ m ⁇ m -2 s -1 .
- Two-way ANOVA shows the cellular content of lutein is significantly affected by both the strain and light intensity.
- Figure I 5d shows that zeaxanthin content in all strains increased with light intensity.
- DF I 5 accumulated the highest amount of zeaxanthin, followed by DF I 7, UTEX 2538, DF40 and CCAP 19/30 accumulated the lowest amount.
- Two-way ANOVA analysis shows that the factors of strain and light intensity determined the accumulation of zeaxanthin. Zeaxanthin accumulation was significantly different among strains and at different light intensities. Among the different strains, DF 17 and UTEX 2538 had similar responses in terms of zeaxanthin accumulation.
- the clustering dendrogram of the examined traits for each strain is shown in Figure 16 and depicts graphically several features of note amongst the strains.
- the correlation analysis shows that accumulation of carotenoids is positively correlated with photosynthesis over all light intensities for the D. salina strains (also shown in Figure 17), signifying a role for carotenoids in photoprotection.
- lutein is not correlated closely with the other carotenoids, but correlates more strongly with photosynthesis and respiration. This result suggests an important and not hitherto identified role for lutein in coordinated control of the cellular functions of photosynthesis and respiration in response to changes in light conditions, which is moreover broadly conserved in Dunaliella strains.
- Glycerol which was not expected to change with light intensity, is weakly correlation with the different carotenoids in the Dunaliella strains as anticipated, but also correlates more closely with either photosynthesis or respiration.
- Olmos SJ, Ochoa L, Paniagua-Michel J, Contreras R (2009) DNA fingerprinting differentiation between ⁇ - carotene hyperproducer strains of Dunaliella from around the world. Saline Systems. 5:5. Olmos-Soto J, Paniagua-Michel J, Contreras PR, Trujillo L (2002) Molecular identification of ⁇ -carotene hyper-456 producing strains of Dunaliella from saline environments using species-specific oligonucleotides. Biotech Lett. 24: 365-369.
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CN109880745A (en) * | 2019-03-15 | 2019-06-14 | 江苏大学 | A method of using pickling waste water, shining bittern water subsection filter salt algae |
CN111518699A (en) * | 2020-05-19 | 2020-08-11 | 唐山市银海食盐有限公司 | High salinity-resistant dunaliella salina, preparation method and application thereof in preparation of purified seawater and sea salt |
CN116158448A (en) * | 2023-02-27 | 2023-05-26 | 清华大学深圳国际研究生院 | Application of marine microalgae in improving plant stress resistance and seaweed fertilizer |
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Non-Patent Citations (3)
Title |
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M. GARCÍA-GONZÁLEZ ET AL: "Conditions for open-air outdoor culture of Dunaliella salina in southern Spain", JOURNAL OF APPLIED PHYCOLOGY., vol. 15, no. 2/3, 1 March 2003 (2003-03-01), NL, pages 177 - 184, XP055474343, ISSN: 0921-8971, DOI: 10.1023/A:1023892520443 * |
OLMOS JORGE ET AL: "DNA fingerprinting differentiation between Î-carotene hyperproducer strains of Dunaliella from around the world", SALINE SYSTEMS, BIOMED CENTRAL, LONDON, GB, vol. 5, no. 1, 30 June 2009 (2009-06-30), pages 5, XP021059921, ISSN: 1746-1448, DOI: 10.1186/1746-1448-5-5 * |
XU YANAN ET AL: "The influence of photoperiod and light intensity on the growth and photosynthesis ofDunaliella salina(chlorophyta) CCAP 19/30", PLANT PHYSIOLOGY AND BIOCHEMISTRY, GAUTHIER-VILLARS, PARIS, FR, vol. 106, 17 May 2016 (2016-05-17), pages 305 - 315, XP029673811, ISSN: 0981-9428, DOI: 10.1016/J.PLAPHY.2016.05.021 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109880745A (en) * | 2019-03-15 | 2019-06-14 | 江苏大学 | A method of using pickling waste water, shining bittern water subsection filter salt algae |
CN111518699A (en) * | 2020-05-19 | 2020-08-11 | 唐山市银海食盐有限公司 | High salinity-resistant dunaliella salina, preparation method and application thereof in preparation of purified seawater and sea salt |
CN111518699B (en) * | 2020-05-19 | 2023-08-22 | 唐山市银海食盐有限公司 | High-salinity-tolerance brine alga, preparation method and application thereof in preparation of purified seawater and sea salt |
CN116158448A (en) * | 2023-02-27 | 2023-05-26 | 清华大学深圳国际研究生院 | Application of marine microalgae in improving plant stress resistance and seaweed fertilizer |
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