botryococcus braunii ktzing ( 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 ; 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 . 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 ( hubner 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 ) . 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 in japan , b. braunii is found over a wide geographical range across the japanese islands ( unpublished data ) .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 . , we demonstrate that b. braunii cells could survive dehydrating conditions for over 6  months and that desiccation - treated cells gained tolerance to extreme temperatures . 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 . 2004 ) at 22  c under a 12-h light/12-h dark cycle with white fluorescent illumination ( approximately 100  mol  photons  m  s ) . b. braunii cultures ( 500  l ) were transferred to a 1.5-ml plastic tube . the tube was placed with its lid open in the growth chamber at 22  c . the cell viability after 6- , 8- , and 12-month desiccation was examined using a growth test . to stain polysaccharides , 3  l of a crystal violet solution ( 10  mg  ml in methanol ) was added to a 100-l stationary - phase culture ( tanoi et al . 2013 ) . after 2  weeks of desiccation , the tubes containing the desiccation - treated or untreated ( control ) cultures ( 500  l containing ca . c in the dark for 1 , 4 , 10 , and 20  days . cell viability was then examined using the growth test . to examine cell survival after desiccation treatment with or without subsequent exposure to an extreme temperature cells in each tube were suspended in 1  ml af-6 medium , and 30-l aliquots containing ca . 75 colonies each were then dispensed into 32 wells of a 96-well plastic plate containing 100  l 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  mol  photons  m  s ) , each well was observed for b. braunii growth under an inverted light microscope . 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 . 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 . ( n  =  3 ) tolerance of b. braunii to long - term desiccation . bar = s.d . ( n  =  3 ) 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 . whole colonies ( a , b ) and enlarged views ( c , d ) are shown for bod - ng17 ( a , c ) and bod - gj2 ( b , d ) . bars indicate 50 and 10  m in the upper and lower micrographs , respectively visualization of extracellular polysaccharide arrays by crystal violet staining . whole colonies ( a , b ) and enlarged views ( c , d ) are shown for bod - ng17 ( a , c ) and bod - gj2 ( b , d ) . bars indicate 50 and 10  m in the upper and lower micrographs , respectively 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 . 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 . 3increased tolerance of desiccation - treated cells of b. braunii to high temperature and freezing . ( n  =  3 ) increased tolerance of desiccation - treated cells of b. braunii to high temperature and freezing . 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 . the extensive extracellular matrices of b. braunii colonies seemed to be associated with the desiccation tolerance . in the present study , extracellular polysaccharides protect algal cells against desiccation by preventing cellular water loss ( cameron 1962 ; clegg 2001 ; oren 2007 ; sakamoto et al . 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 . 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 . ( 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 nostoc 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 . ( c ) flexibility of the cell walls is critical for the desiccation tolerance of an aeroterrestrial green alga klebsormidium crenulatum ( ktz . ) lokhorst ( holzinger et al . 2011 ) and similar mechanisms may be in action in b. braunii . the dehydrated cells of both bod - ng17 and bod - gj2 gained tolerance to high ( 40  c ) and freezing ( 20  c ) temperatures . 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 . 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 .