Abstract
Botryococcus braunii Kützing, a green colonial microalga, occurs worldwide in both freshwater and brackish water environments. Despite considerable attention to B. braunii as a potential source of renewable fuel, many ecophysiological properties of this alga remain unknown. Here, we examined the desiccation and temperature tolerances of B. braunii using two newly isolated strains BOD-NG17 and BOD-GJ2. Both strains survived through 6- and 8-month desiccation treatments but not through a 12-month treatment. Interestingly, the desiccation-treated cells of B. braunii gained tolerance to extreme temperature shifts, i.e., high temperature (40 °C) and freezing (−20 °C). Both strains survived for at least 4 and 10 days at 40 and −20 °C, respectively, while the untreated cells barely survived at these temperatures. These traits would enable long-distance dispersal of B. braunii cells and may account for the worldwide distribution of this algal species. Extracellular substances such as polysaccharides and hydrocarbons seem to confer the desiccation tolerance.
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Introduction
Botryococcus braunii Kützing (Trebouxiophyceae, Chlorophyta), a green colonial microalga, is a cosmopolitan species that occurs worldwide in both freshwater and brackish water environments and robustly produces hydrocarbons (Banerjee et al. 2002). The hydrocarbon content of B. braunii colonies is much higher than that of any other oil-producing microalga and can reach up to 75 % of the dry weight (Chisti 2007). Therefore, B. braunii has received considerable attention as a potential source of renewable fuels. Recent studies have drastically advanced our understanding of the biochemical, molecular biological, and applied aspects of this organism (Baba et al. 2012; Demura et al. 2012; Ioki et al. 2012a, b, c, d; Magota et al. 2012; Matsushima et al. 2012; Molnar et al. 2012; Niehaus et al. 2011; Ranga Rao et al. 2012; Shiho et al. 2012; Xu et al. 2011, 2012; Yonezawa et al. 2012). However, several basic ecophysiological properties of this alga related to its worldwide occurrence still remain unknown.
The desiccation tolerance of microalgae has been previously reported in terrestrial cyanobacteria (Cameron 1962; Dodds et al. 1995; Sakamoto et al. 2009), desert green algae (Gray et al. 2007), phototrophic biofilms algae (Häubner et al. 2006; Rindi 2007; Gustavs et al. 2010), and alpine soil algae (Karsten et al. 2010; Holzinger et al. 2011). Desert biological soil crusts contain many unicellular green algae belonging to three major classes, i.e., Chlorophyceae, Trebouxiophyceae, and Charophyceae (Lewis and Flechtner 2002). Gustavs et al. (2010) found five trebouxiophytes in aeroterrestrial biofilms. Trebouxiophycean algae have also been detected in the air as “aeroalgae” (Handa et al. 2007). Because most of these algae are distributed worldwide, desiccation tolerance appears to be a key ecophysiological trait pertaining to the dispersal of microalgal species on a global scale.
B. braunii is widely distributed in the freshwater environments of Europe, Africa, Asia, Australia, North America, and South America (Aaronson et al. 1983). In Japan, B. braunii is found over a wide geographical range across the Japanese islands (unpublished data). Phylogenetic analysis of 31 B. braunii strains collected from various localities in Japan failed to detect any reliable correlation between phylogeny and localities, suggesting that B. braunii is dispersed between ponds, presumably by wind or birds (Kawachi et al. 2012). However, desiccation tolerance of B. braunii is still unknown. In this study, we demonstrate that B. braunii cells could survive dehydrating conditions for over 6 months and that desiccation-treated cells gained tolerance to extreme temperatures.
Materials and methods
Botryococcus braunii colonies were isolated from freshwater bodies in the Okinawa Prefecture of Japan using a micropipette. Two strains, BOD-NG17 and BOD-GJ2, were established in a unialgal culture and a clonal state. The 18S rDNA sequences of these strains, which we used for species confirmation, were deposited in the GenBank/EMBL/DDBJ database with following accession numbers: AB758446 and AB758447 for BOD-NG17 and BOD-GJ2, respectively. These strains were maintained in test tubes containing AF-6 medium (Kasai et al. 2004) at 22 °C under a 12-h light/12-h dark cycle with white fluorescent illumination (approximately 100 μmol photons m−2 s−1).
Desiccation treatment
B. braunii cultures (500 μL) were transferred to a 1.5-mL plastic tube. Each tube contained ca. 2,500 colonies for both strains. The tube was placed with its lid open in the growth chamber at 22 °C. The culture medium evaporated within 2 weeks. The cell viability after 6-, 8-, and 12-month desiccation was examined using a growth test.
Histochemical staining of polysaccharides
To stain polysaccharides, 3 μL of a crystal violet solution (10 mg mL−1 in methanol) was added to a 100-μL stationary-phase culture (Tanoi et al. 2013). The stained cells were photographed using a microscope equipped with a digital camera.
High temperature and freezing treatment
After 2 weeks of desiccation, the tubes containing the desiccation-treated or untreated (control) cultures (500 μL containing ca. 2,500 colonies) were incubated at 40 or −20 °C in the dark for 1, 4, 10, and 20 days. Cell viability was then examined using the growth test.
The growth test
To examine cell survival after desiccation treatment with or without subsequent exposure to an extreme temperature, growth tests were performed using three independent tubes for each measurement. Cells in each tube were suspended in 1 mL AF-6 medium, and 30-μL aliquots containing ca. 75 colonies each were then dispensed into 32 wells of a 96-well plastic plate containing 100 μL each of AF-6 medium. After incubation for 1 month at 22 °C under a 12-h light/12-h dark cycle with white fluorescent illumination (approximately 100 μmol photons m−2 s−1), each well was observed for B. braunii growth under an inverted light microscope. Wells showing more than a fivefold increase in the colony number were counted as “wells with viable B. braunii” and their percentage was calculated for each tube. This method allowed us to compare culture viability between the treatments because it was otherwise difficult to estimate the cell viability due to the tightly packed colonies of B. braunii.
Results
Desiccation tolerance
Both BOD-NG17 and BOD-GJ2 remained viable under dehydrating conditions for at least 8 months. After 6 months of desiccation, the percentages of wells with growing B. braunii cells reached 100 % in the growth tests for both strains. After 8 months of desiccation, the percentages decreased to 13.5 ± 11.8 % (n = 3) and 3.1 ± 2.1 % (n = 3) for BOD-NG17 and BOD-GJ2, respectively. For both strains, no growth of the rehydrated cells was detected after 12 months of desiccation (Fig. 1).
Extracellular polysaccharide arrays
Histochemical staining of polysaccharides in BOD-NG17 and BOD-GJ2 revealed that the cells of both strains are surrounded by radially oriented arrays of polysaccharides. BOD-GJ2 exhibited sparser arrays than BOD-NG17 (Fig. 2).
Acquired tolerance of desiccation-treated cells to high temperature and freezing
Extreme temperatures damage B. braunii cells. Incubation of nondesiccation-treated BOD-NG17 or BOD-GJ2 cells at 40 °C for only 1 day completely inhibited growth. Incubation of BOD-NG17 and BOD-GJ2 at −20 °C for 10 and 4 days, respectively, resulted in no growth recovery after thawing. However, B. braunii desiccation-treated cells survived under these extreme temperatures. Desiccation-treated cultures of both strains remained viable at 40 °C for at least 4 days. After incubation at 40 °C for 1 day, the percentages of wells with growing B. braunii cells reached 100 and 47.9 ± 21.5 % (n = 3) in the growth tests of desiccation-treated BOD-NG17 and BOD-GJ2, respectively. The percentages decreased to 21.9 ± 8.3 % (n = 3) and 8.3 ± 2.8 % (n = 3) after incubation of BOD-NG17 and BOD-GJ2, respectively, at 40 °C for 4 days. For both strains, no growth was detected after incubation at 40 °C for 10 and 20 days. At −20 °C, desiccation-treated cultures of both strains survived for at least 10 days but no longer than 20 days. For BOD-NG17, the percentage of wells with growing B. braunii cells in the growth tests were 100 %, 100 %, 28.3 ± 6.3 % (n = 3), and 0 % following 1-, 4-, 10-, and 20-day incubation, respectively. For BOD-GJ2, the percentages were 58.3 ± 38.9 % (n = 3), 6.3 ± 2.1 % (n = 3), 7.3 ± 3.5 % (n = 3), and 0 % following 1-, 4-, 10-, and 20-day incubation (Fig. 3).
Discussion
In this study, we determined the culture viability, rather than the cell viability, after desiccation and extreme temperature treatments because the tendency of B. braunii to form tightly packed colonies made estimation of the cell viability difficult. Effects of different treatments were successfully evaluated using a growth test that determined the culture viability based on the percentage of wells containing growing B. braunii. Both BOD-NG17 and BOD-GJ2 exhibited tolerance to desiccation for over 6 months. This is the first report on desiccation tolerance of B. braunii.
The extensive extracellular matrices of B. braunii colonies seemed to be associated with the desiccation tolerance. In the present study, histochemical staining revealed that B. braunii colonies are surrounded by radial arrays of polysaccharides. Extracellular polysaccharides protect algal cells against desiccation by preventing cellular water loss (Cameron 1962; Clegg 2001; Oren 2007; Sakamoto et al. 2009; Tamaru et al. 2005). The radial arrays of polysaccharides were more abundant in BOD-NG17 than in BOD-GJ2. Robustness of the polysaccharide arrays may be associated with relatively high desiccation tolerance of BOD-NG17. In addition, the extracellular matrix also retains a large quantity of liquid hydrocarbons and a network of cross-linked hydrocarbons, and each B. braunii cell is surrounded by a cup-shaped “retaining wall” outside its cell wall (Weiss et al. 2012; Wolf 1983). The retaining walls and hydrocarbons may guard the B. braunii cell from various stresses.
Cellular events associated with the desiccation tolerance of B. braunii are presently ambiguous. Speculations include (a) involvement of sugar alcohols (polyols) like sorbitol and ribitol as in the case of aeroterrestrial trebouxiophytes such as Stichococcus sp., Coccomyxa sp., Chlorella spp., and Apatococcus lobatus (Chodat) J.B. Petersen (Gustavs et al. 2010). (b) Various saccharides, such as arabinose, galactose, fucose, glucose, mannose, and deoxyhexoses, have been detected from B. braunii (Banerjee et al. 2002; Metzger et al. 1990; Weiss et al. 2012) and they also might be involved in anhydrobiosis. In N ostoc commune Vaucher, physiological activities including trehalose accumulation change under desiccation (Fukuda et al. 2008; Tamaru et al. 2005; Sakamoto et al. 2009; Reina-Bueno et al. 2012). (c) Flexibility of the cell walls is critical for the desiccation tolerance of an aeroterrestrial green alga Klebsormidium crenulatum (Kütz.) Lokhorst (Holzinger et al. 2011) and similar mechanisms may be in action in B. braunii. Physical characteristics of B. braunii cell walls need to be clarified.
The dehydrated cells of both BOD-NG17 and BOD-GJ2 gained tolerance to high (40 °C) and freezing (−20 °C) temperatures. However, the underlying mechanisms remain unclear. It is possible that polyols are involved because they act not only as osmolytes, but also as antioxidants, heat protectants, and rapidly available respiratory substrates (Gustavs et al. 2010).
In conclusion, B. braunii is tolerant to desiccation and the dehydrated cells gain tolerance to extreme temperatures. The tolerance of B. braunii to desiccation and extreme temperatures, which algal cells often encounter when carried by birds or wind, enables its dispersal on a global scale.
References
Aaronson S, Berner T, Gold K, Kushner N, Patni NJ, Repak RD (1983) Some observations on the green planktonic alga, Botryococcus braunii and its bloom form. J Plankton Res 5:693–700
Baba M, Ioki M, Nakajima N, Shiraiwa Y, Watanabe MM (2012) Transcriptome analysis of an oil-rich race A strain of Botryococcus braunii (BOT-88-2) by de novo assembly of pyrosequencing cDNA reads. Bioresour Technol 109:282–286
Banerjee A, Sharma R, Chisti Y, Banerjee UC (2002) Botryococcus braunii: a renewable source of hydrocarbons and other chemicals. Crit Rev Biotechnol 22:245–279
Cameron RE (1962) Species of Nostoc vaucher occurring in the Sonoran Desert in Arizona. Trans Am Microsc Soc 81:379–384
Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306
Clegg JS (2001) Cryptobiosis—a peculiar state of biological organization. Comp Biochem Physiol B Biochem Mol Biol 128:613–624
Demura M, Kawachi M, Koshikawa H, Nakayama T, Mayuzumi Y, Watanabe MM (2012) Succession of genetic diversity of Botryococcus braunii (Trebouxiophyceae) in two Japanese reservoirs. Proc Environ Sci 15:3–11
Dodds WK, Gudder DA, Mollenhauer D (1995) The ecology of Nostoc. J Phycol 31:2–18
Fukuda SY, Yamakawa R, Hirai M, Kashino Y, Koike H, Satoh K (2008) Mechanisms to avoid photoinhibition in a desiccation-tolerant cyanobacterium, Nostoc commune. Plant Cell Physiol 49:488–492
Gray DW, Lewis LA, Cardon ZG (2007) Photosynthetic recovery following desiccation of desert green algae (Chlorophyta) and their aquatic relatives. Plant Cell Environ 30:1240–1255
Gustavs L, Eggert A, Michalik D, Karsten U (2010) Physiological and biochemical responses of green microalgae from different habitats to osmotic and matric stress. Protoplasma 243:3–14
Handa S, Ohmura Y, Nakano T, Nakahara-Tsubota M (2007) Airborne green microalgae (Chlorophyta) in snowfall. Hikobia 15:109–120
Häubner N, Schumann R, Karsten U (2006) Aeroterrestrial microalgae growing in biofilms on facades—response to temperature and water stress. Microb Ecol 51:285–293
Holzinger A, Lütz C, Karsten U (2011) Desiccation stress causes structural and ultrastructural alterations in the aeroterrestrial green alga Klebsormidium crenulatum (Klebsormidiophyceae, Streptophyta) isolated from an alpine soil crust. J Phycol 47:591–602
Ioki M, Baba M, Bidadi H, Suzuki I, Shiraiwa Y, Watanabe MM, Nakajima N (2012a) Modes of hydrocarbon oil biosynthesis revealed by comparative gene expression analysis for race A and race B strains of Botryococcus braunii. Bioresour Technol 109:271–276
Ioki M, Baba M, Nakajima N, Shiraiwa Y, Watanabe MM (2012b) Transcriptome analysis of an oil-rich race B strain of Botryococcus braunii (BOT-22) by de novo assembly of pyrosequencing cDNA reads. Bioresour Technol 109:292–296
Ioki M, Baba M, Nakajima N, Shiraiwa Y, Watanabe MM (2012c) Transcriptome analysis of an oil-rich race B strain of Botryococcus braunii (BOT-70) by de novo assembly of 5′-end sequences of full-length cDNA clones. Bioresour Technol 109:277–281
Ioki M, Ohkoshi M, Nakajima N, Nakahira-Yanaka Y, Watanabe MM (2012d) Isolation of herbicide-resistant mutants of Botryococcus braunii. Bioresour Technol 109:300–303
Karsten U, Lütz C, Holzinger A (2010) Ecophysiological performance of the aeroterrestrial green alga Klebsormidium crenulatum (Charophyceae, Streptophyta) isolated from an alpine soil crust with an emphasis on desiccation stress. J Phycol 46:1187–1197
Kasai F, Kawachi M, Erata M, Watanabe MM (2004) NIES collection list of strains, 7th edn. National Institute for Environmental Studies, Tsukuba, p 49
Kawachi M, Tanoi T, Demura M, Kaya K, Watanabe MM (2012) Relationship between hydrocarbons and molecular phylogeny of Botryococcus braunii. Algal Res 1:114–119
Lewis LA, Flechtner VR (2002) Green algae (Chlorophyta) of desert microbiotic crusts: diversity of North American taxa. Taxon 51:443–451
Magota A, Saga K, Okada S, Atobe S, Imou K (2012) Effect of thermal pretreatments on hydrocarbon recovery from Botryococcus braunii. Bioresour Technol 123:195–198
Matsushima D, Jenke-Kodama H, Sato Y, Fukunaga Y, Sumimoto K, Kuzuyama T, Matsunaga S, Okada S (2012) The single cellular green microalga Botryococcus braunii, race B possesses three distinct 1-deoxy-D-xylulose 5-phosphate synthases. Plant Sci 185–186:309–320
Metzger P, Allard B, Casadevall E, Berkaloff C, Coute A (1990) Structure and chemistry of a new chemical race of Botryococcus braunii (Chlorophyceae) that produces lycopadiene, a tetraterpenoid hydrocarbon. J Phycol 26:258–266
Molnar I, Lopez D, Wisecaver JH, Devarenne TP, Weiss TL, Pellegrini M, Hackett JD (2012) Bio-crude transcriptomics: gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa). BMC Genomics 13(1):576
Niehaus TD, Okada S, Devarenne TP, Watt DS, Sviripa V, Chappell J (2011) Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii. Proc Natl Acad Sci U S A 108:12260–12265
Oren A (2007) Diversity of organic osmotic compounds and osmotic adaptation in cyanobacteria and algae. In: Seckbach J (ed) Algae and cyanobacteria in extreme environments. Springer, Berlin, pp 641–655
Ranga Rao A, Ravishankar GA, Sarada R (2012) Cultivation of green alga Botryococcus braunii in raceway, circular ponds under outdoor conditions and its growth, hydrocarbon production. Bioresour Technol 123:528–533
Reina-Bueno M, Arganodona M, Salvador M, Rodriguez-Moya J, Iglesias-Guerra F, Csonka LN, Nieto JJ, Vargas C (2012) Role of trehalose in salinity and temperature tolerance in the model halophilic bacterium Chromohalobacter salexigens. PLoS One 7(3):e33587
Rindi F (2007) Diversity, distribution and ecology of green algae and cyanobacteria in urban habitats. In: Seckbach J (ed) Algae and cyanobacteria in extreme environments. Springer, Berlin, p 619–655
Sakamoto T, Yoshida T, Arima H, Hatanaka Y, Takani Y, Tamaru Y (2009) Accumulation of trehalose in response to desiccation and salt stress in the terrestrial cyanobacterium. Phycol Res 57:66–73
Shiho M, Kawachi M, Horioka K, Nishita Y, Ohashi K, Kaya K, Watanabe MM (2012) Business evaluation of a green microalgae Botryococcus braunii oil production system. Proc Environ Sci 15:90–109
Tamaru Y, Takani Y, Yoshida T, Sakamoto T (2005) Crucial role of extracellular polysaccharides in desiccation and freezing tolerance in the terrestrial cyanobacterium Nostoc commune. Appl Environ Microbiol 71:7327–7333
Tanoi T, Kawachi M, Watanabe MM (2013) Appearance of fibrillary exopolysaccharides secreted by Botryococcus braunii. Phycol Res, in press
Weiss TL, Roth R, Goodson C, Vitha S, Black I, Azadi P, Rusch J, Holzenburg A, Devarenne TP, Goodenough U (2012) Colony organization in the green alga Botryococcus braunii (Race B) is specified by a complex extracellular matrix. Eukaryot Cell 11:1424–1440
Wolf FR (1983) Botryococcus braunii an unusual hydrocarbon-producing alga. Appl Biochem Biotech 8:249–260
Xu L, Guo C, Wang F, Zheng S, Liu CZ (2011) A simple and rapid harvesting method for microalgae by in situ magnetic separation. Bioresour Technol 102:10047–10051
Xu L, Liu R, Wang F, Liu CZ (2012) Development of a draft-tube airlift bioreactor for Botryococcus braunii with an optimized inner structure using computational fluid dynamics. Bioresour Technol 119:300–305
Yonezawa N, Matsuura H, Shiho M, Kaya K, Watanabe MM (2012) Effects of soybean curd wastewater on the growth and hydrocarbon production of Botryococcus braunii strain BOT-22. Bioresour Technol 109:304–307
Acknowledgments
This work was supported by the Core Research for Evolutionary Science and Technology program of Japan Science and Technology Agency and the Project for the Practical Application of Energy from Algal Biomass of the Tsukuba International Strategic Comprehensive Special Zone.
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Demura, M., Ioki, M., Kawachi, M. et al. Desiccation tolerance of Botryococcus braunii (Trebouxiophyceae, Chlorophyta) and extreme temperature tolerance of dehydrated cells. J Appl Phycol 26, 49–53 (2014). https://doi.org/10.1007/s10811-013-0059-7
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DOI: https://doi.org/10.1007/s10811-013-0059-7