http://dx.doi.org/10.4490/algae.2013.28.2.185 Open Access
Cold-tolerant strain of Haematococcus pluvialis (Haematococcaceae, Chlorophyta) from Blomstrandhalvøya (Svalbard)
Tatyana A. Klochkova
1, Min Seok Kwak
2, Jong Won Han
2, Taizo Motomura
3, Chikako Nagasato
3and Gwang Hoon Kim
2,*
1Kamchatka State Technical University (KamchatGTU), Petropavlovsk-Kamchatsky, Klyuchevskaya 35, 683003, Russia
2Department of Biology, Kongju National University, Kongju 314-701, Korea
3Muroran Marine Station, Field Science Centre for Northern Biosphere, Hokkaido University, Muroran 051-0003, Japan
A new cold-adapted Arctic strain of Haematococcus pluvialis from Blomstrandhalvøya Island (Svalbard) is described.
This strain is predominantly always in non-motile palmelloid stage. Transmission electron microscopy showed the pres- ence of very thick cell wall and abundant lipid vesicles in the palmelloids, including red and green cells. The external morphology of the non-motile palmelloid and motile bi-flagellated cells of our strain is similar to H. pluvialis; however it differs from H. pluvialis in physiology. Our strain is adapted to live and produce astaxanthin in the low temperature (4- 10°C), whilst the usual growth temperature for H. pluvialis is between 20-27°C. Phylogenetic analysis based on 18S rRNA gene data showed that our strain nested within the Haematococcus group, forming a sister relationship to H. lacustris and H. pluvialis, which are considered synonymous. Therefore, we identified our Arctic strain as H. pluvialis.
Key Words: astaxanthin; Blomstrandhalvøya; cold resistance; Haematococcus pluvialis; Svalbard; transmission electron microscopy
INTRODUCTION
The genus Haematococcus (Chlorophyceae, Volvo- cales) is cosmopolitan, reported from all continents ex- cept Antarctica (Guiry and Guiry 2013). Seven species of Haematococcus have been currently accepted taxonomi- cally and among them, H. pluvialis Flotow is the best studied. This ubiquitous microalga occurs primarily in principally ephemeral small fresh water pools in temper- ate climate, where it passes through the life cycle in as few as 4 days (e.g., Droop 1954, Czygan 1970, Thompson and Wujek 1989). It is well known for the ability to synthesize and accumulate esters of the red ketocarotenoid astax- anthin (3,39-dihydroxy-b,b-carotene-4,49-dione) for up to 4% of the total cellular dry weight under laboratory-
induced stress conditions, such as nutrient deprivation, increased salinity, high irradiance, and exposure to high temperature (30-33°C), as well as combinations of these stresses (summarized in Collins et al. 2011). In natural environment, accumulation of the astaxanthin is an ad- aptation to habitats that exhibit strong radiation, in addi- tion to the formation of cysts having rigid cell walls (e.g., Hagen et al. 1994, 2002, Montsant et al. 2001).
Astaxanthin is generally known to be used as a food coloring agent, natural feed additive for the poultry in- dustry and for aquaculture, especially as a feed supple- ment in the culture of salmon, trout and shrimp, and for medicinal and cosmetic application due to its powerful
Received April 15, 2013, Accepted May 18, 2013
*
Corresponding Author E-mail: ghkim@kongju.ac.krTel: +82-41-850-8504, Fax: +82-41-850-8479 This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://cre- ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
with an argon-krypton laser using a 488-nm excitation line and AOBS filter-free system collecting emitted light between 498 and 700 nm. A series of optical sections of chloroplast was captured and used for 3D reconstruction of morphology. The autofluorescence of the chlorophyll was exploited for visualization of chloroplast structure.
Electron microscopic observations
Three fixation methods were applied to fix cells in vegetative (green) and aplanospore (red) stages, includ- ing chemical fixation, cryofixation, and high-pressure freezing. The chemical fixation and embedding methods were performed as described by Klochkova et al. (2006), and the cryofixation and embedding methods were per- formed as described by Terauchi et al. (2012). For high pressure freezing and freeze substitution, a pellet of cells was placed between two copper planchets and rap- idly frozen in a high-pressure freezing instrument (EM PACT2; Leica, Solms, Germany) at a pressure of approxi- mately 2,000 atm and at the temperature of liquid nitro- gen. The samples were then placed in liquid nitrogen, and the top of the copper tube was peeled away for further processing. The following cryofixation and embedding methods were performed as described by Terauchi et al.
(2012). Resin-embedded samples were sectioned with a diamond knife, and thin sections stained with 4% uranyl acetate for 30 min and lead citrate (Reynolds 1963) for 10 min were viewed and photographed on a Hitachi H-300 transmission electron microscope (TEM) (Hitachi, Tokyo, Japan).
Molecular phylogenetic analysis
The DNA of H. pluvialis was extracted using an Invi- sorb Spin Plant Mini Kit (Invitek, Berlin-Buch, Germany) according to the manufacturer’s protocol. Extracted DNA was stored at -20°C and used for the amplification of 18S rRNA. The 18S rRNA was amplified and sequenced using the following primers: G01 (5′-CACCTGGTTGATCCTGC- CAG-3′), G14 (5′-CCTTGGCAGACGCTTTCGCAG-3′), G04 (5′-CAGAGGTGAAATTCTTGGAT-3′), and G07 (5′-GCTT- GATCCTTCTGCAGGTTCACCTAC-3′) (Saunders et al.
1995). Polymerase chain reaction and sequencing were performed as detailed in Klochkova et al. (2006, 2008). All sequences of the forward and reverse strands were deter- mined and the electropherograms were edited using the program Chromas v.1.45 (McCarthy 1998). Nucleotide sequences were aligned using Se-Al v2.0a11 (Rambaut 2002).
antioxidant capacity. Most of the astaxanthin used for aquaculture is synthetically derived; however, growing demand exists for commercial production of astaxan- thin from natural sources. Therefore, numerous studies regarding Haematococcus focused on the astaxanthin- producing properties of H. pluvialis, using strains mainly from culture collections (i.e., The Culture Collection of Al- gae at the University of Texas at Austin [UTEX], National Institute for Environmental Studies, Japan [NIES], Cul- ture Collection of Algae and Protozoa [CCAP], The Global Bioresource Center [ATCC], Culture Collection of Algae at Goettingen [SAG], Algae Culture Collection at Norsk Institutt for Vannforskning [NIVA]) and grown under a wide range of culture conditions (González et al. 2009).
The usual growth temperature for H. pluvialis is between 20-27°C.
We recently found a new cold-adapted Arctic strain of Haematococcus on Blomstrandhalvøya Island (Sval- bard) (Kim et al. 2011). Our strain, tentatively named as Haematococcus sp. (Kim et al. 2011), exhibits growth be- tween 4-15°C, whereas the average summer temperature of Svalbard reaches 4-6°C, and January averages at -12°C to -16°C. The purpose of the present study was to provide more morphological and physiological details for this pe- culiar Arctic strain of Haematococcus and to address its phylogenetic relationships to other species in the genus.
MATERIALS AND METHODS Sample collection and culture
Specimen of Haematococcus pluvialis was collected on Blomstrandhalvøya Island (78°59′ N, 12°03′ E) in a small freshwater basin in the rock fed with melted snow by the authors G. H. K., T. A. K., and J. W. H. (Kim et al. 2011).
The unialgal strain was isolated as described by Kim et al. (2011) and grown in a modified liquid ATCC Medium 625 (Klochkova et al. 2006), Bold’s basal medium (Bischoff and Bold 1963), and conventional BG11 medium in 90 × 60-mm plastic Petri dishes and 150-mL glass flasks at 4-15°C, 30-35 µmol photons m-2 s-1 provided by cool- white fluorescent lighting and 12 : 12 h light : dark regime.
Micrographs were taken with Olympus DP50 digital cam- era (Olympus, Tokyo, Japan) affixed to an Olympus BX50 microscope using Viewfinder Lite and Studio Lite com- puter programs.
For investigation of chloroplast morphology, cells were observed under an Olympus Fluoview laser scanning confocal microscope. The microscope was equipped
(AB360747; isolate NIES-144, collected from Sapporo, Hokkaido) and Haematococcus sp. (FJ877140; origin un- known). The clade included H. pluvialis (AF159369; iso- late SAG 34-1b, collected from former Czechoslovakia), although it had less affinity to our strain. Species H. lacus- tris has been synonymized with H. pluvialis. If they are conspecific, our strain positioning between them should also be attributed to H. pluvialis until more information is available on various strains of the H. pluvialis-lacustris complex.
Morphology
Our strain is predominantly always in non-motile palmelloid stage. Both green- and red-colored spheri- cal non-motile palmelloid cells (Fig. 2A, B & G) are 19.2- 44.8 µm in diameter (28.2 ± 1.9 µm on average) and have a thick cell wall (secondary wall) reaching up to 930 nm thick (Fig. 3A). Some cells are ellipsoidal. The centrally Phylogeny of 18S rRNA was reconstructed using maxi-
mum likelihood (ML). ML analyses were performed with RAxML v.7.2.8 (Stamatakis 2006) using the GTRGAMMA model. We used 300 independent tree inferences, apply- ing options of automatically optimized surface plasmon resonance (SPR) rearrangement and 25 distinct rate cat- egories in the program to identify the best tree. Statistical support for each branch was obtained by 1,000 bootstrap replications with the same substitution model. The se- quence determined in this study has been deposited in GenBank under accession number KC986379.
RESULTS
Molecular phylogeny
Our strain nested within the Haematococcus group (Fig. 1), forming a sister relationship to H. lacustris
18S rRNA
0.01 Chlamydomonas pulsatilla AF514404
Carteria radiosa AF182819
Haematococcus lacustris AB360747
Protosiphon botryoides U41177
Chloromonas subdivisa AF517080
Haematococcus pluvialis (Svalbard) KC986379
Dunaliella parva M6299 Stephanosphaera sp. AB360751
Characium vacuolatum M63001
Pteromonas protracta X91627 Chlamydomonas monadina AY220559
Phacotus lenticularis X91628 Haematococcus sp. FJ877140
Haematococcus pluvialis AF159369
Scenedesmus obliquus EF564131
Chlorococcum ellipsoideum U70586 Haematococcus zimbabwiensis U70797
Pleurastrum insigne Z28972
Dunaliella salina DQ009779 72
88 98
87
98 14
100 100
100 100
99 100
78 94
98
100
Fig. 1.
Maximum likelihood tree estimated from 18S rRNA sequence data. Numbers above the branches indicate bootstrap values.F) of 200-1,000 nm in diameter. The cytoplasm was het- erogeneous and segregated (Fig. 3E); however this might have been an artifact of the fixation.
Motile bi-flagellated cells (Fig. 2E) were rarely seen in field-collected samples (4 bi-flagellated cells among several hundred non-motile astaxanthin-containing red cells). Also, they are rarely seen in the laboratory culture.
Motile spores have a cup-shaped chloroplast with 1-2 pyrenoids and one large vacuole positioned in the cell center or several smaller scattered vacuoles. Cytoplasmic threads extending from the protoplast to the wall are ex- tremely fine. The cells retain motility for 1 day after re- lease, and thereafter they settle on the substrate and be- gin to develop palmelloid morphology.
Growth temperature
When this strain was first brought from the field, it could live only at 4-6°C; however >80% of the cells were always red-colored. The cells show slow growth at 4-6°C.
positioned nucleus was up to 9.5 µm in size, with a large nucleolus (data not shown), and a small Golgi body posi- tioned next to the nucleus was observed (Fig. 3G). Large chloroplast with numerous starch grains occupies the whole cell’s periphery (Figs 2C, D, 3B & C). Chloroplast of young cells contains 2-4 pyrenoids and large old cells have up to 8-12 pyrenoids. No lamellae enter the pyrenoid and the starch sheath is composed of numerous grains (Fig.
3B & D). The thylakoids are stacked in a rather random manner (Fig. 3B-D). Numerous small mitochondria were mostly located beneath the cell membrane (Fig. 3A) and some mitochondria were seen in the cytoplasm (Fig. 3C).
Lipid vesicles packed almost the whole cytoplasmic volume in the cells. Massive lipid accumulation occurred regardless of the pigment accumulation, since green- colored palmelloids (Fig. 2A & B) also contained numer- ous lipid vesicles (Fig. 3C). In the green palmelloids lipid vesicles were less electron-dense and 200-400 nm in di- ameter, whereas red palmelloids (i.e., astaxanthin-con- taining) had more electron-dense lipid vesicles (Fig. 3E &
Fig. 2.
Light microscopy of Haematococcus pluvialis cells. (A & B) Through-focus images of the same green palmelloid cell cultured at 15°C, showing chloroplast with several pyrenoids occupying almost the whole cell’s periphery (arrows). (C & D) Morphology of large parietal chloroplast seen with confocal microscopy. (C) Reconstruction of the chloroplast shape by overlaying serial sections. (D) Section taken through the cell’s center, showing lack of chlorophyll autofluorescence in the center and its restricted localization to the cells’ periphery. (E) Motile bi-flagellated cell in 20°C. (F) Red pigmentation due to astaxanthin accumulation appears towards the center of palmelloid cells in 6°C (arrowheads). (G) Crimson- colored astaxanthin-containing palmelloid cell developed in 6°C. Scale bars represent: A-G, 10 μm.A B C D
E F G
Fig. 3.
Electron microscopy of Haematococcus pluvialis palmelloid cells. (A) Thick cell wall of the palmelloid cell, showing trilaminar sheath (tls, arrow) and smooth secondary wall (sw). Arrowheads point to mitochondria located beneath the cell membrane. (B) Arrangement of the thylakoids in the chloroplast. (C) Numerous lipid vesicles (asterisks) scattered in the cytoplasm of green palmelloid cell. (D) Pyrenoid within a starch-containing chloroplast. (E) Electron-dense astaxanthin-containing lipid vesicles (asterisks) scattered in the heterogeneous segregated cytoplasm (arrowheads) of the red palmelloids. (F) Enlarged lipid vesicle. Arrows point to a membrane surrounding the vesicle. (G) Vertical section through the Golgi body (g). Arrows point to the adjacent Golgi vesicles. ecm, extracellular matrix; is, interspace; m, mitochondrion; p, pyrenoid; s, starch; t, thylakoids. Scale bars represent: A, C & E-G, 200 nm; B & D, 500 nm.A
C D
B
E F G
backs in its vegetative culture to increase the growth rate and culture productivity.
Knowledge of the morphology and life cycle of H.
pluvialis becomes increasingly important due to the in- terest in this organism as a biotechnological source of astaxanthin. However, studies on its morphogenesis and ultrastructure proceed slowly in comparison to other microalgae. The palmelloid cells have so thick cell wall that chemical fixation and embedding prior to ultrathin sectioning and electron microscopy becomes almost im- possible (Hagen et al. 2002). The cells of our strain were notoriously difficult to fix for TEM using chemical fixa- tion method and cryofixation. These treatments resulted in turgor breakage and complete disintegration of the cellular contents, and formation of massive ice crystals rupturing the organelles, respectively. Only high-pressure freezing with the following cryofixation was partially suc- cessful to preserve some cell structures more or less in- tact. Our TEM results were conclusive with Hagen et al.
(2002), showing presence of thick and smooth secondary wall, which is believed to be mainly composed of granu- lose non-fibrillar mannan I, a trilaminar sheath, and a layer of extracellular matrix in the palmelloid cells. The cytoplasm of the green palmelloids was packed with lipid vesicles, implying that abundant lipid accumulation oc- curs prior to the accumulation of the astaxanthin inside the vesicles.
Phylogenetic relationships within Haematococcus have been poorly resolved and only four members of the genus have been sequenced to date, including H. droebakensis Wollenweber, H. lacustris (Girod-Chantrans) Roststafin- ski, H. pluvialis, and H. zimbabwiensis Pockock, result- ing in 1, 11, 211, and 6 nucleotide sequences of different products for each species deposited in the GenBank, respectively (National Center for Biotechnology Infor- mation 2013). Also, nucleotide sequences of different products for 3 unidentified Haematococcus sp. have been deposited in the GenBank (National Center for Biotech- nology Information 2013). Although H. pluvialis is the most often used for sequencing, no comprehensive study was undertaken to resolve relationships among different strains registered in the database. The name H. pluvialis might have been commonly attached to any red-colored palmelloid Haematococcus, while different similar look- ing isolates might potentially be numerous new cryptic species. For instance, species H. lacustris is also synony- mized with H. pluvialis (e.g., Hagen et al. 2002, Pentecost 2002), however they have a principal morphological dif- ference such as H. lacustris is predominantly always in motile flagellated stage (Pocock 1960), whereas H. plu- After a period of 860 days in culture at this temperature,
we counted only 230,000-866,000 cells in a total volume of 20 mL of BG11 medium, which were progeny from 1 cell.
Over the subsequent period of laboratory culture for 1.5 years, this isolate was gradually acclimated to grow at 15°C. When cultured at 15°C under 30-35 µmol photons m-2 s-1, palmelloids are mostly green-colored. This strain reproduces efficiently by means of motile flagellated spores after 1-2 days-long exposure to 20°C; however it is not able to live at this temperature for more than 2-3 days. Astaxanthin is accumulated by stressing cells grown at 15°C under 30-35 mol photons m-2 s-1 with low tem- perature between 4-10°C under 30-35 µmol photons m-2 s-1. Under cold stress, efficient astaxanthin production occurs even in the low light (approximately 1.7-9 µmol photons m-2 s-1). Red globular regions appear towards the center of palmelloid cells (Fig. 2F), and after several days of the same treatment cells appear completely red (Fig.
2G).
DISCUSSION
The cold-tolerant strain of Haematococcus pluvialis discussed in the present study was collected only from one site on Blomstrandhalvøya Island, which was a small rock pool located at a distance of several meters from the sea, but we did not find it in other surveyed areas on this island and in the vicinity of Ny-Ǻlesund (Kim et al. 2008, 2011). Droop (1961) also noted that H. pluvialis typically inhabited rock pools, often, although not nec- essarily, within a few feet of the sea. We previously sug- gested anthropogenic introduction of some microalgae to this Arctic area (Kim et al. 2011). It is noteworthy that no Haematococcus microalgae were recorded from this region before and in general H. pluvialis inhabits temper- ate climate. When defining area of species distribution one should consider that it is limited by the temperature for maximum and / or optimum reproduction and not by the temperatures for survival. Our strain of H. pluvialis from Blomstrandhalvøya reproduces efficiently at 15- 20°C, while the average summer temperature of Svalbard reaches 4-6°C. If introduction supposedly took place, it is not a recent event and should have occurred at least a number of years ago, since the cells had to go through several generations of their life cycle to adapt to the harsh Arctic environment. The unfavorable environment might have influenced the specifically low growth rate of this strain. Further work is required to overcome the draw-
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Guiry, M. D. & Guiry, G. M. 2013. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. Available from: http://www.algaebase.org. Ac- cessed Mar 27, 2013.
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Kim, G. H., Klochkova, T. A., Han, J. W., Kang, S. -H., Choi, H.
G., Chung, K. W. & Kim, S. J. 2011. Freshwater and ter- restrial algae from Ny-Ålesund and Blomstrandhalvøya Island (Svalbard). Arctic 64:25-31.
Kim, G. H., Klochkova, T. A. & Kang, S. -H. 2008. Notes on freshwater and terrestrial algae from Ny-Ålesund, Sval- bard (high Arctic sea area). J. Environ. Biol. 29:485-491.
Klochkova, T. A., Cho, G. -Y., Boo, S. M., Chung, K. W., Kim, S.
J. & Kim, G. H. 2008. Interactions between marine facul- tative epiphyte Chlamydomonas sp. (Chlamydomonad- ales, Chlorophyta) and ceramiaceaen algae (Rhodophy- ta). J. Environ. Biol. 29:427-435.
Klochkova, T. A., Kang, S. -H., Cho, G. Y., Pueschel, C. M., West, J. A. & Kim, G. H. 2006. Biology of a terrestrial green alga, Chlorococcum sp. (Chlorococcales, Chlo- rophyta), collected from the Miruksazi stupa in Korea.
Phycologia 45:349-358.
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au/chromas.html. Accessed Dec 7, 2012.
Montsant, A., Zarka, A. & Boussiba, S. 2001. Presence of a nonhydrolyzable biopolymer in cell wall of vegetative cells and astaxanthin-rich cysts of Haematococcus plu- vialis (Chlorophyceae). Mar. Biotechnol. 3:515-521.
National Center for Biotechnology Information. 2013. Gen- Bank. Available from: http://www.ncbi.nlm.nih.gov. Ac- cessed Mar 27, 2013.
Pentecost, A. 2002. Order Volvocales. In John, D. M., Whitton, B. A. & Brook, A. J. (Eds.) The Freshwater Algal Flora of the British Isles: An Identification Guide to Freshwater and Terrestrial Algae. Cambridge University Press, Cam- vialis is predominantly always in non-motile palmelloid
stage. Until more comprehensive phylogenetic analysis on different strains of H. pluvialis from around the world is complete, we suggest attributing our strain to this spe- cies based on current phylogenetic result. Morphologi- cally, it is not very different from H. pluvialis to support its separation as a distinct species; however it significantly differs in physiology. Unlike other described strains of H.
pluvialis, our strain is adapted to live and produce astax- anthin in the low temperature (4-10°C). Most commonly cultured species of algae are able to grow at temperatures between 16 and 27°C and the optimal growth tempera- tures are usually in the range of 18-20°C (Chen et al. 2009).
As evident from the numerous references on H. pluvialis, its usual growth temperature for commercial cultivation is between 20-27°C. Our psychrophilic strain is therefore an exception and can potentially be used for astaxanthin exploitation in colder climates.
ACKNOWLEDGEMENTS
We express our sincere thanks to Dr. G. C. Zuccarello for his help with the phylogenetic analysis of Haemato- coccus. This research was a part of the project entitled
‘Long-term change of structure and function in marine ecosystems of Korea’ funded by the Ministry of Oceans and Fisheries, Korea.
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