Perspectives of green microalgal research for biofuels

Jianhua Liu

Ocean Research Center of Zhoushan Zhejiang University Zhoushan ZJ 316021 P R China and Ocean College Zhejiang University Hangzhou ZJ 310058 P R China 0013066zjueducn

Abstract Green microalgae (Chlorophytes) are thought to be the most effi cient organisms for biofuel production However their potential has not been fully realized Large-scale production of algal biofuels is not sustainable with current algal strains and cultivation technologies In this manuscript I discuss the rationale of research approaches focused on domestication that we are about to take and some major issues need to be resolved for algal biofuel production

Keywords Green microalgae breeding biofuels functional genomics synthetic biology

Rapid economic growth is tightly associated with high energy consumption leading to the increase of fuel prices greenhouse gas emissions and environment impacts Additionally it accelerates the depletion of fossil fuel reserves (Chisti 2008) Hence there is an urgent need for renewable and sustainable alternative fuels to alleviate the rapid consumption of fossil fuels While wind turbines and solar panels are get-ting popular for generating electricity biofuels have received a renewed interest in li-quid fuel production for transportation and aviation that heavily replies on petroleum-based fuels (Wijffels amp Barbosa 2010)

Microalgae are thought to be the most effi cient crop for biofuel or diesel produc-tion when compared to soybean camelina sunfl ower jatropha and oil palm (Ferrell amp Sarisky-Reed 2010) Moreover it would not compete with food crops for arable land and fresh water (Chisti 2008 Ferrell amp Sarisky-Reed 2010 Wijffels amp Barbosa 2010) In addition it sequesters carbon dioxide a major type of greenhouse gases and thus mitigates global warming However current naturally occurring microalgal spe-cies suffer from poor genetic traits such as slow growth rate low cell density and low lipid contents resembling precursors of modern crops (Hu et al 2008)

Commercial cultivation of microalgae such as Spirulina Chlorella Dunaliella and Haematococcus is largely limited to products of human nutrient supplements at a relatively small scale (eg 10ndash100 tonsyear) (Benemann 2008) The total yield (ie

Algological Studies 145146 (2014) p 15ndash25 ArticlePublished online February 2014

copy E Schweizerbartrsquosche Verlagsbuchhandlung Stuttgart Germany DOI 1011271864-131820140154

wwwschweizerbartde1864-13180154 $ 300

eschweizerbart_xxx

16 Jianhua Liu

dry weight) of these microalgae is about 10000 tonsyear half of which is produced in China However cultivation of microalgae for biofuels is not commercially viable now In 2001 GreenFuel Technologies Corporation was set up at Cambridge MT USA they developed a process of growing algae using emissions from power plant to produce biofuels from algae The company expanded in 2005 and closed in 2009 due to the unexpected high cost of algal fuel production (Kanellos 2009) According to John Benemann the algal industry has suffered from ldquofantastic promotions bizarre cultivation systems and absurd productivity projectionsrdquo Why are we not drowning in algal biofuels now The answer remains the same as what in 90rsquos algal biofuels are expensive compared to fossil fuels (ie US$ 300ndash400 per gallon versus US$ 90ndash100 per gallon) (Gabel 2012)

Recently a committee on the sustainable development of algal biofuels in USA has expressed concerns on sustainability of the scale-up of algal biofuel production (Hunter-Cevera 2012) Based on their review it has been concluded that production of 39 billion liters of algae-based biofuels would place unsustainable demands on ener-gy water and nutrients with current technologies and requires much more knowledge (Hunter-Cevera 2012) The energy outputs of algal biofuels by some systems are less than the energy inputs for producing the fuels That is the energy return on investment (EROI) is less than 1 (Sills et al 2013)

Nevertheless the committee has acknowledged that algal biofuels have the po-tential to contribute to improving the sustainability of the transportation sector and additional innovations that require research and development are needed to realize the full potential of algal biofuels (Hunter-Cevera 2012 Rhode 2012) particularly in the following 4 points (1) Algal strain selection and improvement to enhance desired characteristics and biofuel productivity (2) An EROI that is comparable to other transportation fuels or at least improving and approaching the EROIs of other transportation fuels (3) The use of wastewater for cultivating algae for fuels or the recycling of harvest water particularly if freshwater algae are used and (4) Recycling of nutrients in algal biofuel pathways that require harvesting unless co-products that meet an equivalent nutrient need are produced

This manuscript includes a brief review on genomic analysis of green microalgae tools available for genetic and molecular analyses and the approach for exploring the potential for algal biofuel production especially in the green microalgae (Chloro-phytes)

Genomic and transcriptomic analysis of green microalgae

According to the genome online database GOLD (wwwgenomeonlineorg) 4430 genome sequencing projects have been completed so far and 22539 genome sequenc-ing projects have gotten underway However most of them are relatively small bacte-rial genomes Up to now only a handful of green microalgal (Chlorophyta) genome sequencing projects have been completed They include Chlamydomonas reinhardtii

eschweizerbart_xxx

Perspectives of green microalgal research for biofuels 17

(Merchant et al 2007) Chlorella variabilis (Blanc et al 2010) Volvox carteri (Proch-nik et al 2010) Micromonas pusilla (Worden et al 2009) and Ostreococcus spp (Palenik et al 2007) Nonetheless these genomes have already provided a tremen-dous amount of information relating to chloroplasts and the structure assembly and function of eukaryotic fl agella (cilia) (Merchant et al 2007) virus-algal interaction (Blanc et al 2010) evolution of multicellularity and development (Prochnik et al 2010) green evolution and dynamic adaptations (Worden et al 2009) and the smallest eukaryotes (Palenik et al 2007)

Next-generation sequencing (NGS) technologies (Church 2006 Hall 2007) complemented by paired-end library of long inserts (Ruan amp Wei 2010) and the de Bruijn graph-based sequence-alignment algorithms (Kundeti et al 2010 Compeau et al 2011) enable cheap and quick assembly of draft genomes without cosmid or BAC clones Draft genomes contain relatively small (eg lt10Kb) non-redundant as-semblies or contigs without order To produce a fi nished genome from a draft genome contigs need to be orientated in a sequential order and gaps need to be fi lled Draft genomes are cost-effective and useful for a variety of studies because most genes are represented in the draft sequence More and more (permanent) draft genomes will be released by the DOE-Joint Genome Institute (JGI) USA (httpgenomejgidoegovgenome-projects)

Transcriptional profi ling analyses of microalgae in response to various stress can provide information related to gene functions and transcription regulatory networks (Smith 2012) Microarray technologies require genome sequence information On the other hand NGS technologies coupled with de Bruijn graph-based algorithms permit effi cient assembly of transcriptomes without reference genome Profi ling analyses based on de novo assembled transcriptomes have been reported in a number of non-model green microalgae such as Dunaliella tertiolecta (Rismani-Yazdi et al 2011) Neochloris oleoabundans (Rismani-Yazdi et al 2012) Botryococcus braunii (Baba et al 2012 Ioki et al 2012) and Botryosphaerella sudeticus (Sun et al 2013) Mining these transcriptional data would allow identifi cation of genes and metabolic pathways essential for growth and lipid biosynthesis

Tools for genetic and molecular analyses in green microalgae

Experimental evaluation of biological function of novel candidate genes discovered from mining of genomic and transcriptomic datasets requires tools for DNA mani-pulation in microalgae C reinhardtii is the best studied green microalga for which many DNA manipulation tools have been established for example genetic crossing between complementary mating types mt+ and mtndash (Harris 1998) transformation of exogenous DNA using glass-bead agitation electroporation or particle bombardment with various selective marker genes (Debuchy et al 1989 Kindle et al 1989 Diener et al 1990 Mayfi eld amp Kindle 1990 Shimogawara et al 1998) insertional mutagene-sis for easy identifi cation of tagged alleles (Tam amp Lefebvre 1993 Davies et al 1994

eschweizerbart_xxx

18 Jianhua Liu

Davies et al 1996 Smith amp Lefebvre 1996 Koutoulis et al 1997 Smith amp Lefebvre 1997 Asleson amp Lefebvre 1998 Wykoff et al 1998 Davies et al 1999 Gonzalez-Ballester et al 2011) and gene knock-down with antisense or siRNA for evaluation of gene function (Zhao amp Yu 2008 Kim amp Cerutti 2009) RNA interference-mediated gene silencing mechanisms are useful for knock-down of a gene of interest (Ibrahim et al 2006 Zhao et al 2007) However it also affects the expression of exogenous genes without selection in C reinhardtii To circumvent this expression of polycis-tronic RNA containing the internal ribosome entry site (IRES) or polycistronic pro-tein containing an intra-ribosomal cleavage signal peptide 2A for stable expression of non-selective genes has been established in C reinhardtii (Manuell amp Mayfi eld 2006 Rasala et al 2012)

Targeted gene modifi cation would be achieved if a double-strand break (DSB) could be created at a right locus in genome By fusion a DNA-binding zinc-fi nger domain with an endonuclease domain Kim et al have shown for the fi rst time that a hybrid restriction enzyme can generate a sequence-specifi c DSB in genome (Kim et al 1996) Recently this zinc-fi nger nuclease (ZFN) approach has been successfully employed in C reinhardtii (Sizova et al 2013) On the other hand DNA transformation would be one of the hurdles for genome manipulation Nano-sized polymer particles such as guanidinium-rich molecular transporters (GR-MoTrs) (Wender et al 2000 Hyman et al 2012) and stearic acid grafted chitosan oligosaccharide (CSO-SA) (Hu et al 2006) have shown to be able to enhance delivery of small molecules in mammalian cell-line systems Recently enhancing DNA transformation by GR-MoTrs has been success-fully employed in microalgal species including C reinhardtii N oleoabundans and Scenedesmus dimorphus (Hyman et al 2012) The nano-sized polymer particle-based transformation approach will allow genome manipulation in non-model algae

Methodologies for promoting contents of TAG and other molecules in green microalgae

C reinhardtii has also been used as a model for biofuel studies Triacylglycerol (TAG) and starch contents accumulate in C reinhardtii upon nutrient or nitrogen depletion (Wang et al 2009 Moellering amp Benning 2010 Work et al 2010) It has been found that disruption of starch storage enhances TAG storage increasing productivity of biofuels (Wang et al 2009 Work et al 2010) Cell density of C reinhardtii cultures under phototrophic conditions is relatively low and unsuitable for commercial bio-mass production

D salina is one of the best studied industrial green microalgal species that is cur-rently cultivated at large scale for commercial production of beta-carotenes (Bene-mann 2008) Like C reinhardtii it undergoes a vegetative and a sexual life cycle (Polle amp Qin 2009) However genetic crosses have not been established in laboratory Transformations of exogenous DNA using glass-bead agitation electroporation and particle bombardment with various selection marker genes have been reported (Porath

eschweizerbart_xxx

Perspectives of green microalgal research for biofuels 19

et al 1997 Jin et al 2001 Geng et al 2003 Tan et al 2005) Genome sequencing project of D salina is under way at the JGI

B braunii is well known for its high hydrocarbons content as much as 50ndash80 of dry cell weight (DCW) (Banerjee et al 2002 Metzger amp Largeau 2005) B braunii is believed to be the major contributor to fossil fuel deposits (Weiss et al 2010 Niehaus et al 2011) Unlike TAG in other microalgae hydrocarbons in B braunii resemble the chemicals found in petroleum and can be directly processed with the current in-frastructures of petroleum industry However B braunii suffers extreme slow growth rate and is unsuitable for molecular manipulation or large-scale production

Chlorella spp were thought to be the super-food due to its high content of proteins ie up to 45 of DCW in late 1940s and early 1950s (Belasco 1997) Nowadays the interest has been focused on its lipid content for biofuels A number of studies have shown that lipid contents in Chlorella spp can reach as high as 40 of DCW under phototrophic outdoor growth conditions (Tang et al 2011 Zhou et al 2013) Yet the maximal density of the phototrophic cultures is just a few grams DCW per liter (Tang et al 2011 Zhou et al 2013) Though the maximal cell density of heterotrophic cultures of Chlorella spp is very high compared to that of phototrophic ones (ie ~100 g L versus ~3 g L DCW) (Wu amp Shi 2007 Doucha amp Livansky 2012) demand for organic carbon in heterotrophic growth would limit its applications for large-scale production of low-cost biofuels

Scenedesmus spp is also widely used for studies of algae-based biofuels because of its ease in cultivation and adaptation to a wide range of environmental conditions (Chinnasamy et al 2010 Ho et al 2010 Pan et al 2011 Xia et al 2013) These algae are likely suitable feedstock for biofuel production among others

Current problems associated with biofuel production from microalgae

Due to a severe food shortage at the time during the period from late 1940 and early 1950 after WWII green microalgae such as Chlorella spp were seen as a potential source for human nutrient or super-food (Belasco 1997) Recently because of the soaring petroleum price scientists have renewed their interest in developing algal feedstock for biofuels to alleviate the demand on petroleum (Chisti 2008) However it is now realized that microalgae are much more diffi cult to produce than previously thought and production of algae-based biofuels is not economically viable (Hu et al 2008 Wijffels amp Barbosa 2010)

Major issues in algal feedstock production for biofuels are poor genetic traits such as slow growth low cell density and low lipid content of the current microalgal spe-cies (Hu et al 2008 Wijffels amp Barbosa 2010) Modern crops such as corns have undergone extensive domestication from their ancient precursors (Flint-Garcia 2013) Clearly domestication of the current algal species is necessary for large-scale produc-tion of algae-based biofuels in the economically viable and environmentally sustain-able way

eschweizerbart_xxx

20 Jianhua Liu

Future research to unlock the biofuel potential of green microalgae

Biofuel potential of microalgae has not been fully realized up to today though they were proposed for biomass production in photobioreactors nearly 40 years ago (Per-soone amp Sorgeloos 1975) Up to now it is said that production of algae-based biofuels at a billion liter-scale is unsustainable regarding usage of energy water and nutrients (Benemann 2008 Hunter-Cevera 2012) We believe that the biggest issue is the slow growth rate and low cell density in cultivation of microalgae under phototrophic con-ditions Then to what extend is the growth rate and cell density of algal cultures a reality As a matter of fact nature has provided an answer to us ndash the algal blooms

Microalgal blooms a massive occurrence of algae near the surface of lake river or sea occurs occasionally (Wake amp Hillen 1980 Aaronson et al 1983 Landsberg 2002) It is believed that certain environmental conditions trigger algal blooms Ac-cordingly the algae must be able to sense the environmental cues of blooms We hypothesize that the environmental cues trigger the probable bloom-sensing signaling pathway that in turn activates cellular metabolisms for rapid growth alters cell-cell communication (to-be-revealed) for high cell-density and secure nutrients by secret-ing toxins or allomones known as harmful algal blooms (Landsberg 2002)

Unfortunately it is not possible to maintain rapid growth rates and high cell densi-ties of algae collected from sites of blooms in the laboratory (Aaronson et al 1983) This could be due to the change of growth conditions Alternatively blooms may re-present a unique algal growth phase resembling the cancerous phase in multi-cellular organisms a terminal stage of growth Cells enter the blooming state or rapid growth phase might not be able to return to its initial phase of slow growth rate and low cell density This would impose a great challenge on study of genes involved in bloom signaling and regulation of growth rate and cell density during algal blooms

Assuming activation of the bloom-signaling cascades by mutation could initiate the rapid growth phase under nutrient replete conditions in absence of environmen-tal cues one would obtain the rapidly growing algal mutant strains through genetic screening One of the bloom inducers a bloom inducer gene 1 (or BIG1) has been isolated from the diatom Thalassiosira pseudonana and has been patented (Mock amp Hipkin 2012) However if algae after blooming are unable to return to the regular life cycle and cease to grow non-tagged mutations induced by physical or chemical mutagens would not be suitable for the analysis On the other hand because blooming strains would dominate over others in a pool of mutations tagged mutations gener-ated via DNA insertion could provide information about genes involved in sensing environmental cues for bloom and regulation of rapid growth and high cell-density Though these alleles would not be viable after blooming conditional alleles could resolve the issue

To test this hypothesis one will need to effi ciently generate a large number of insertional mutations A pool of mutations can be grown and maintained by subcultur-ing Rapidly growing strains will have predominant patterns of RFLP by insertion

eschweizerbart_xxx

Perspectives of green microalgal research for biofuels 21

Hence the RFLP pattern of subcultures will tell if a blooming strain(s) appeared Insertional alleles can be readily identifi ed using various methods such as TAIL-PCR plasmid rescue or single-stranded DNA ligation methodologies (Zhang amp Chiang 1996 Singer amp Burke 2003 Coll 2006) By using this approach genes involved in regulation of rapid growth and high cell density can be identifi ed Construction of conditional alleles will be necessary for construction of rapidly growing algae if blooming algae cannot re-enter their life cycles

Targeting nuclear gene manipulation is not possible in most green microalgae though siRNA-mediated gene knock-down is widely used in C reinhardtii (Zhao amp Yu 2008 Kim amp Cerutti 2009) Homologous recombination-mediated gene knockout turns out to be possible in a non-green microalga Nannochloropsis sp (Chromalveolata) (Kilian et al 2011) Recently targeting gene modifi cation using the bacterial immune CRISPR CAS system has been a great success in mammalian fi sh and fl y (Cong et al 2013 Gratz et al 2013 Hwang et al 2013 Mali et al 2013 Wang et al 2013) Estab-lishing the CRISPR CAS system in green algae would permit evaluating function of genes involved in regulation of algal blooms and construct conditional alleles that are readily induced to blooms for effi cient production of biomass This approach is also ap-plicable for isolation of algal strains resistant to environmental stress factors

Acknowledgements

I would like to thank colleagues at the Ocean Research Centre of Zhoushan and Ocean College Zhejiang University for technical and administrative assistances and the Zhoushan City local government for fi nancial support

References

Aaronson S Berner T Gold K Kushner L Patni NJ Repak A amp Rubin D (1983) Some observations on the green planktonic alga Botryococcus braunii and its bloom form ndash J Plank-ton Res 5 693ndash700

Asleson CM amp Lefebvre PA (1998) Genetic analysis of fl agellar length control in Chlamydomonas reinhardtii a new long-fl agella locus and extragenic suppressor mutations ndash Genetics 148 693ndash702

Baba M Ioki M Nakajima N Shiraiwa Y amp Watanabe MM (2012) Transcriptome analysis of an oil-rich race A strain of Botryococcus braunii (BOT-88-2) by de novo assembly of pyrose-quencing cDNA reads ndash Bioresour Technol 109 282ndash286

Banerjee A Sharma R Chisti Y amp Banerjee UC (2002) Botryococcus braunii a renewable source of hydrocarbons and other chemicals ndash Crit Rev Biotechnol 22 245ndash279

Belasco W (1997) Algae burgers for a hungry world The rise and fall of chlorella cuisine ndash Tech-nology and Culture 38 608ndash634

Benemann JR (2008) Opportunities and challenges in algae biofuel production httpwwwfutureenergyeventscomalgae

Blanc G Duncan G Agarkova I Borodovsky M Gurnon J Kuo A Lindquist E Lucas S et al (2010) The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis coevolution with viruses and cryptic sex ndash Plant Cell 22 2943ndash2955

eschweizerbart_xxx

Perspectives of green microalgal research for biofuels 19

et al 1997 Jin et al 2001 Geng et al 2003 Tan et al 2005) Genome sequencing project of D salina is under way at the JGI

B braunii is well known for its high hydrocarbons content as much as 50ndash80 of dry cell weight (DCW) (Banerjee et al 2002 Metzger amp Largeau 2005) B braunii is believed to be the major contributor to fossil fuel deposits (Weiss et al 2010 Niehaus et al 2011) Unlike TAG in other microalgae hydrocarbons in B braunii resemble the chemicals found in petroleum and can be directly processed with the current in-frastructures of petroleum industry However B braunii suffers extreme slow growth rate and is unsuitable for molecular manipulation or large-scale production

Chlorella spp were thought to be the super-food due to its high content of proteins ie up to 45 of DCW in late 1940s and early 1950s (Belasco 1997) Nowadays the interest has been focused on its lipid content for biofuels A number of studies have shown that lipid contents in Chlorella spp can reach as high as 40 of DCW under phototrophic outdoor growth conditions (Tang et al 2011 Zhou et al 2013) Yet the maximal density of the phototrophic cultures is just a few grams DCW per liter (Tang et al 2011 Zhou et al 2013) Though the maximal cell density of heterotrophic cultures of Chlorella spp is very high compared to that of phototrophic ones (ie ~100 g L versus ~3 g L DCW) (Wu amp Shi 2007 Doucha amp Livansky 2012) demand for organic carbon in heterotrophic growth would limit its applications for large-scale production of low-cost biofuels

Scenedesmus spp is also widely used for studies of algae-based biofuels because of its ease in cultivation and adaptation to a wide range of environmental conditions (Chinnasamy et al 2010 Ho et al 2010 Pan et al 2011 Xia et al 2013) These algae are likely suitable feedstock for biofuel production among others

Current problems associated with biofuel production from microalgae

Due to a severe food shortage at the time during the period from late 1940 and early 1950 after WWII green microalgae such as Chlorella spp were seen as a potential source for human nutrient or super-food (Belasco 1997) Recently because of the soaring petroleum price scientists have renewed their interest in developing algal feedstock for biofuels to alleviate the demand on petroleum (Chisti 2008) However it is now realized that microalgae are much more diffi cult to produce than previously thought and production of algae-based biofuels is not economically viable (Hu et al 2008 Wijffels amp Barbosa 2010)

Major issues in algal feedstock production for biofuels are poor genetic traits such as slow growth low cell density and low lipid content of the current microalgal spe-cies (Hu et al 2008 Wijffels amp Barbosa 2010) Modern crops such as corns have undergone extensive domestication from their ancient precursors (Flint-Garcia 2013) Clearly domestication of the current algal species is necessary for large-scale produc-tion of algae-based biofuels in the economically viable and environmentally sustain-able way

eschweizerbart_xxx

20 Jianhua Liu

Future research to unlock the biofuel potential of green microalgae

Biofuel potential of microalgae has not been fully realized up to today though they were proposed for biomass production in photobioreactors nearly 40 years ago (Per-soone amp Sorgeloos 1975) Up to now it is said that production of algae-based biofuels at a billion liter-scale is unsustainable regarding usage of energy water and nutrients (Benemann 2008 Hunter-Cevera 2012) We believe that the biggest issue is the slow growth rate and low cell density in cultivation of microalgae under phototrophic con-ditions Then to what extend is the growth rate and cell density of algal cultures a reality As a matter of fact nature has provided an answer to us ndash the algal blooms

Microalgal blooms a massive occurrence of algae near the surface of lake river or sea occurs occasionally (Wake amp Hillen 1980 Aaronson et al 1983 Landsberg 2002) It is believed that certain environmental conditions trigger algal blooms Ac-cordingly the algae must be able to sense the environmental cues of blooms We hypothesize that the environmental cues trigger the probable bloom-sensing signaling pathway that in turn activates cellular metabolisms for rapid growth alters cell-cell communication (to-be-revealed) for high cell-density and secure nutrients by secret-ing toxins or allomones known as harmful algal blooms (Landsberg 2002)

Unfortunately it is not possible to maintain rapid growth rates and high cell densi-ties of algae collected from sites of blooms in the laboratory (Aaronson et al 1983) This could be due to the change of growth conditions Alternatively blooms may re-present a unique algal growth phase resembling the cancerous phase in multi-cellular organisms a terminal stage of growth Cells enter the blooming state or rapid growth phase might not be able to return to its initial phase of slow growth rate and low cell density This would impose a great challenge on study of genes involved in bloom signaling and regulation of growth rate and cell density during algal blooms

Assuming activation of the bloom-signaling cascades by mutation could initiate the rapid growth phase under nutrient replete conditions in absence of environmen-tal cues one would obtain the rapidly growing algal mutant strains through genetic screening One of the bloom inducers a bloom inducer gene 1 (or BIG1) has been isolated from the diatom Thalassiosira pseudonana and has been patented (Mock amp Hipkin 2012) However if algae after blooming are unable to return to the regular life cycle and cease to grow non-tagged mutations induced by physical or chemical mutagens would not be suitable for the analysis On the other hand because blooming strains would dominate over others in a pool of mutations tagged mutations gener-ated via DNA insertion could provide information about genes involved in sensing environmental cues for bloom and regulation of rapid growth and high cell-density Though these alleles would not be viable after blooming conditional alleles could resolve the issue

To test this hypothesis one will need to effi ciently generate a large number of insertional mutations A pool of mutations can be grown and maintained by subcultur-ing Rapidly growing strains will have predominant patterns of RFLP by insertion

eschweizerbart_xxx

Perspectives of green microalgal research for biofuels 21

Hence the RFLP pattern of subcultures will tell if a blooming strain(s) appeared Insertional alleles can be readily identifi ed using various methods such as TAIL-PCR plasmid rescue or single-stranded DNA ligation methodologies (Zhang amp Chiang 1996 Singer amp Burke 2003 Coll 2006) By using this approach genes involved in regulation of rapid growth and high cell density can be identifi ed Construction of conditional alleles will be necessary for construction of rapidly growing algae if blooming algae cannot re-enter their life cycles

Targeting nuclear gene manipulation is not possible in most green microalgae though siRNA-mediated gene knock-down is widely used in C reinhardtii (Zhao amp Yu 2008 Kim amp Cerutti 2009) Homologous recombination-mediated gene knockout turns out to be possible in a non-green microalga Nannochloropsis sp (Chromalveolata) (Kilian et al 2011) Recently targeting gene modifi cation using the bacterial immune CRISPR CAS system has been a great success in mammalian fi sh and fl y (Cong et al 2013 Gratz et al 2013 Hwang et al 2013 Mali et al 2013 Wang et al 2013) Estab-lishing the CRISPR CAS system in green algae would permit evaluating function of genes involved in regulation of algal blooms and construct conditional alleles that are readily induced to blooms for effi cient production of biomass This approach is also ap-plicable for isolation of algal strains resistant to environmental stress factors

Acknowledgements

I would like to thank colleagues at the Ocean Research Centre of Zhoushan and Ocean College Zhejiang University for technical and administrative assistances and the Zhoushan City local government for fi nancial support

References

Aaronson S Berner T Gold K Kushner L Patni NJ Repak A amp Rubin D (1983) Some observations on the green planktonic alga Botryococcus braunii and its bloom form ndash J Plank-ton Res 5 693ndash700

Asleson CM amp Lefebvre PA (1998) Genetic analysis of fl agellar length control in Chlamydomonas reinhardtii a new long-fl agella locus and extragenic suppressor mutations ndash Genetics 148 693ndash702

Baba M Ioki M Nakajima N Shiraiwa Y amp Watanabe MM (2012) Transcriptome analysis of an oil-rich race A strain of Botryococcus braunii (BOT-88-2) by de novo assembly of pyrose-quencing cDNA reads ndash Bioresour Technol 109 282ndash286

Banerjee A Sharma R Chisti Y amp Banerjee UC (2002) Botryococcus braunii a renewable source of hydrocarbons and other chemicals ndash Crit Rev Biotechnol 22 245ndash279

Belasco W (1997) Algae burgers for a hungry world The rise and fall of chlorella cuisine ndash Tech-nology and Culture 38 608ndash634

Benemann JR (2008) Opportunities and challenges in algae biofuel production httpwwwfutureenergyeventscomalgae

Blanc G Duncan G Agarkova I Borodovsky M Gurnon J Kuo A Lindquist E Lucas S et al (2010) The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis coevolution with viruses and cryptic sex ndash Plant Cell 22 2943ndash2955

eschweizerbart_xxx

20 Jianhua Liu

Future research to unlock the biofuel potential of green microalgae

Biofuel potential of microalgae has not been fully realized up to today though they were proposed for biomass production in photobioreactors nearly 40 years ago (Per-soone amp Sorgeloos 1975) Up to now it is said that production of algae-based biofuels at a billion liter-scale is unsustainable regarding usage of energy water and nutrients (Benemann 2008 Hunter-Cevera 2012) We believe that the biggest issue is the slow growth rate and low cell density in cultivation of microalgae under phototrophic con-ditions Then to what extend is the growth rate and cell density of algal cultures a reality As a matter of fact nature has provided an answer to us ndash the algal blooms

Microalgal blooms a massive occurrence of algae near the surface of lake river or sea occurs occasionally (Wake amp Hillen 1980 Aaronson et al 1983 Landsberg 2002) It is believed that certain environmental conditions trigger algal blooms Ac-cordingly the algae must be able to sense the environmental cues of blooms We hypothesize that the environmental cues trigger the probable bloom-sensing signaling pathway that in turn activates cellular metabolisms for rapid growth alters cell-cell communication (to-be-revealed) for high cell-density and secure nutrients by secret-ing toxins or allomones known as harmful algal blooms (Landsberg 2002)

Unfortunately it is not possible to maintain rapid growth rates and high cell densi-ties of algae collected from sites of blooms in the laboratory (Aaronson et al 1983) This could be due to the change of growth conditions Alternatively blooms may re-present a unique algal growth phase resembling the cancerous phase in multi-cellular organisms a terminal stage of growth Cells enter the blooming state or rapid growth phase might not be able to return to its initial phase of slow growth rate and low cell density This would impose a great challenge on study of genes involved in bloom signaling and regulation of growth rate and cell density during algal blooms

Assuming activation of the bloom-signaling cascades by mutation could initiate the rapid growth phase under nutrient replete conditions in absence of environmen-tal cues one would obtain the rapidly growing algal mutant strains through genetic screening One of the bloom inducers a bloom inducer gene 1 (or BIG1) has been isolated from the diatom Thalassiosira pseudonana and has been patented (Mock amp Hipkin 2012) However if algae after blooming are unable to return to the regular life cycle and cease to grow non-tagged mutations induced by physical or chemical mutagens would not be suitable for the analysis On the other hand because blooming strains would dominate over others in a pool of mutations tagged mutations gener-ated via DNA insertion could provide information about genes involved in sensing environmental cues for bloom and regulation of rapid growth and high cell-density Though these alleles would not be viable after blooming conditional alleles could resolve the issue

To test this hypothesis one will need to effi ciently generate a large number of insertional mutations A pool of mutations can be grown and maintained by subcultur-ing Rapidly growing strains will have predominant patterns of RFLP by insertion

eschweizerbart_xxx

Perspectives of green microalgal research for biofuels 21

Hence the RFLP pattern of subcultures will tell if a blooming strain(s) appeared Insertional alleles can be readily identifi ed using various methods such as TAIL-PCR plasmid rescue or single-stranded DNA ligation methodologies (Zhang amp Chiang 1996 Singer amp Burke 2003 Coll 2006) By using this approach genes involved in regulation of rapid growth and high cell density can be identifi ed Construction of conditional alleles will be necessary for construction of rapidly growing algae if blooming algae cannot re-enter their life cycles

Targeting nuclear gene manipulation is not possible in most green microalgae though siRNA-mediated gene knock-down is widely used in C reinhardtii (Zhao amp Yu 2008 Kim amp Cerutti 2009) Homologous recombination-mediated gene knockout turns out to be possible in a non-green microalga Nannochloropsis sp (Chromalveolata) (Kilian et al 2011) Recently targeting gene modifi cation using the bacterial immune CRISPR CAS system has been a great success in mammalian fi sh and fl y (Cong et al 2013 Gratz et al 2013 Hwang et al 2013 Mali et al 2013 Wang et al 2013) Estab-lishing the CRISPR CAS system in green algae would permit evaluating function of genes involved in regulation of algal blooms and construct conditional alleles that are readily induced to blooms for effi cient production of biomass This approach is also ap-plicable for isolation of algal strains resistant to environmental stress factors

Acknowledgements

I would like to thank colleagues at the Ocean Research Centre of Zhoushan and Ocean College Zhejiang University for technical and administrative assistances and the Zhoushan City local government for fi nancial support

References

Aaronson S Berner T Gold K Kushner L Patni NJ Repak A amp Rubin D (1983) Some observations on the green planktonic alga Botryococcus braunii and its bloom form ndash J Plank-ton Res 5 693ndash700

Asleson CM amp Lefebvre PA (1998) Genetic analysis of fl agellar length control in Chlamydomonas reinhardtii a new long-fl agella locus and extragenic suppressor mutations ndash Genetics 148 693ndash702

Baba M Ioki M Nakajima N Shiraiwa Y amp Watanabe MM (2012) Transcriptome analysis of an oil-rich race A strain of Botryococcus braunii (BOT-88-2) by de novo assembly of pyrose-quencing cDNA reads ndash Bioresour Technol 109 282ndash286

Banerjee A Sharma R Chisti Y amp Banerjee UC (2002) Botryococcus braunii a renewable source of hydrocarbons and other chemicals ndash Crit Rev Biotechnol 22 245ndash279

Belasco W (1997) Algae burgers for a hungry world The rise and fall of chlorella cuisine ndash Tech-nology and Culture 38 608ndash634

Benemann JR (2008) Opportunities and challenges in algae biofuel production httpwwwfutureenergyeventscomalgae

Blanc G Duncan G Agarkova I Borodovsky M Gurnon J Kuo A Lindquist E Lucas S et al (2010) The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis coevolution with viruses and cryptic sex ndash Plant Cell 22 2943ndash2955

eschweizerbart_xxx

Perspectives of green microalgal research for biofuels 21

Hence the RFLP pattern of subcultures will tell if a blooming strain(s) appeared Insertional alleles can be readily identifi ed using various methods such as TAIL-PCR plasmid rescue or single-stranded DNA ligation methodologies (Zhang amp Chiang 1996 Singer amp Burke 2003 Coll 2006) By using this approach genes involved in regulation of rapid growth and high cell density can be identifi ed Construction of conditional alleles will be necessary for construction of rapidly growing algae if blooming algae cannot re-enter their life cycles

Targeting nuclear gene manipulation is not possible in most green microalgae though siRNA-mediated gene knock-down is widely used in C reinhardtii (Zhao amp Yu 2008 Kim amp Cerutti 2009) Homologous recombination-mediated gene knockout turns out to be possible in a non-green microalga Nannochloropsis sp (Chromalveolata) (Kilian et al 2011) Recently targeting gene modifi cation using the bacterial immune CRISPR CAS system has been a great success in mammalian fi sh and fl y (Cong et al 2013 Gratz et al 2013 Hwang et al 2013 Mali et al 2013 Wang et al 2013) Estab-lishing the CRISPR CAS system in green algae would permit evaluating function of genes involved in regulation of algal blooms and construct conditional alleles that are readily induced to blooms for effi cient production of biomass This approach is also ap-plicable for isolation of algal strains resistant to environmental stress factors

Acknowledgements

I would like to thank colleagues at the Ocean Research Centre of Zhoushan and Ocean College Zhejiang University for technical and administrative assistances and the Zhoushan City local government for fi nancial support

References

Aaronson S Berner T Gold K Kushner L Patni NJ Repak A amp Rubin D (1983) Some observations on the green planktonic alga Botryococcus braunii and its bloom form ndash J Plank-ton Res 5 693ndash700

Asleson CM amp Lefebvre PA (1998) Genetic analysis of fl agellar length control in Chlamydomonas reinhardtii a new long-fl agella locus and extragenic suppressor mutations ndash Genetics 148 693ndash702

Baba M Ioki M Nakajima N Shiraiwa Y amp Watanabe MM (2012) Transcriptome analysis of an oil-rich race A strain of Botryococcus braunii (BOT-88-2) by de novo assembly of pyrose-quencing cDNA reads ndash Bioresour Technol 109 282ndash286

Banerjee A Sharma R Chisti Y amp Banerjee UC (2002) Botryococcus braunii a renewable source of hydrocarbons and other chemicals ndash Crit Rev Biotechnol 22 245ndash279

Belasco W (1997) Algae burgers for a hungry world The rise and fall of chlorella cuisine ndash Tech-nology and Culture 38 608ndash634

Benemann JR (2008) Opportunities and challenges in algae biofuel production httpwwwfutureenergyeventscomalgae

Blanc G Duncan G Agarkova I Borodovsky M Gurnon J Kuo A Lindquist E Lucas S et al (2010) The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis coevolution with viruses and cryptic sex ndash Plant Cell 22 2943ndash2955

eschweizerbart_xxx

22 Jianhua Liu

Chinnasamy S Bhatnagar A Claxton R amp Das K C (2010) Biomass and bioenergy production potential of microalgae consortium in open and closed bioreactors using untreated carpet indus-try effl uent as growth medium ndash Bioresour Technol 101 6751ndash6760

Chisti Y (2008) Biodiesel from microalgae beats bioethanol ndash Trends Biotechnol 26(3) 126ndash131Church GM (2006) Genomes for all ndash Sci Am 294 46ndash54Coll JM (2006) Methodologies for transferring DNA into eukaryotic microalgae ndash Span J Agric

Res 4 316ndash330Compeau PE Pevzner PA amp Tesler G (2011) How to apply de Bruijn graphs to genome assem-

bly ndash Nat Biotechnol 29 987ndash991Cong L Ran FA Cox D Lin S Barretto R Habib N Hsu PD Wu X et al (2013) Multi-

plex genome engineering using CRISPRCas systems ndash Science 339 819ndash823Davies JP Yildiz FH amp Grossman AR (1994) Mutants of Chlamydomonas with Aberrant re-

sponses to sulfur deprivation ndash Plant Cell 6 53ndash63Davies JP Yildiz FH amp Grossman AR (1996) Sac1 a putative regulator that is critical for sur-

vival of Chlamydomonas reinhardtii during sulfur deprivation ndash EMBO J 15 2150ndash2159Davies JP Yildiz FH amp Grossman AR (1999) Sac3 an Snf1-like serinethreonine kinase that

positively and negatively regulates the responses of Chlamydomonas to sulfur limitation ndash Plant Cell 11 1179ndash1190

Debuchy R Purton S amp Rochaix JD (1989) The argininosuccinate lyase gene of Chlamydomonas reinhardtii an important tool for nuclear transformation and for correlating the genetic and mo-lecular maps of the ARG7 locus ndash EMBO J 8 2803ndash2809

Diener DR Curry AM Johnson KA Williams BD Lefebvre PA Kindle KL amp Rosen-baum JL (1990) Rescue of a paralyzed-fl agella mutant of Chlamydomonas by transformation ndash Proc Natl Acad Sci USA 87 5739ndash5743

Doucha J amp Livansky K (2012) Production of high-density Chlorella culture grown in fermenters ndash J Appl Phycol 24 35ndash43

Ferrell J amp Sarisky-Reed V (2010) National Algal Biofuels Technology Roadmap httpwww1eereenergygovbioenergypdfsalgal_biofuels_roadmappdf

Flint-Garcia SA (2013) Genetics and consequences of crop domestication ndash J Agric Food Chem 61 8267ndash8276

Gabel DA (2012) What is Taking Algae Biofuels So Long Environmental News NetworkGeng DG Wang YQ Wang P Li WB amp Sun YR (2003) Stable expression of hepatitis B

surface antigen gene in Dunaliella salina ndash J Appl Phycol 15 451ndash456Gonzalez-Ballester D Pootakham W Mus F Yang W Catalanotti C Magneschi L de Mon-

taigu A Higuera JJ et al (2011) Reverse genetics in Chlamydomonas a platform for isolating insertional mutants ndash Plant Methods 7 24

Gratz SJ Cummings AM Nguyen JN Hamm DC Donohue LK Harrison MM Wil-donger J amp OrsquoConnor-Giles KM (2013) Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease ndash Genetics 194 1029ndash1035

Hall N (2007) Advanced sequencing technologies and their wider impact in microbiology ndash J Exp Biol 210 1518ndash1525

Harris EH (1998) The Chlamydomonas Sourcebook A Comprehensive Guide to Biology and Laboratory Use San Diego CA USA Academic Press

Ho SH Chen WM amp Chang JS (2010) Scenedesmus obliquus CNW-N as a potential candidate for CO2 mitigation and biodiesel production ndash Bioresour Technol 101 8725ndash8730

Hu FQ Zhao MD Yuan H You J Du YZ amp Zeng S (2006) A novel chitosan oligosaccha-ride-stearic acid micelles for gene delivery properties and in vitro transfection studies ndash Int J Pharm 315 158ndash166

Hu Q Sommerfeld M Jarvis E Ghirardi M Posewitz M Seibert M amp Darzins A (2008) Microalgal triacylglycerols as feedstocks for biofuel production perspectives and advances ndash Plant J 54 621ndash639

eschweizerbart_xxx

Perspectives of green microalgal research for biofuels 23

Hunter-Cevera JC (2012) Sustainable development of algal biofuels Washington DC USA The National Academics Press

Hwang WY Fu Y Reyon D Maeder ML Tsai SQ Sander JD Peterson RT Yeh JR et al (2013) Effi cient genome editing in zebrafi sh using a CRISPR-Cas system ndash Nat Biotechnol 31 227ndash229

Hyman JM Geihe EI Trantow BM Parvin B amp Wender PA (2012) A molecular method for the delivery of small molecules and proteins across the cell wall of algae using molecular trans-porters ndash Proc Nat Acad Sci USA 109 13225ndash13230

Ibrahim F Rohr J Jeong WJ Hesson J amp Cerutti H (2006) Untemplated oligoadenylation promotes degradation of RISC-cleaved transcripts ndash Science 314 1893

Ioki M Baba M Nakajima N Shiraiwa Y amp Watanabe MM (2012) Transcriptome analysis of an oil-rich race B strain of Botryococcus braunii (BOT-22) by de novo assembly of pyrosequenc-ing cDNA reads ndash Bioresour Technol 109 292ndash296

Jin ES Polle JE amp Melis A (2001) Involvement of zeaxanthin and of the Cbr protein in the re-pair of photosystem II from photoinhibition in the green alga Dunaliella salina ndash Biochim Bio-phys Acta 1506 244ndash259

Kanellos M (2009) GreenFuel Technologies Closing Down Greentechmedia Boston MA USAKilian O Benemann CS Niyogi KK amp Vick B (2011) High-effi ciency homologous recombi-

nation in the oil-producing alga Nannochloropsis sp ndash Proc Nat Acad Sci USA 108 21265ndash21269

Kim EJ amp Cerutti H (2009) Targeted gene silencing by RNA interference in Chlamydomonas ndash Methods Cell Biol 93 99ndash110

Kim YG Cha J amp Chandrasegaran S (1996) Hybrid restriction enzymes zinc fi nger fusions to Fok I cleavage domain ndash Proc Nat Acad Sci USA 93 1156ndash1160

Kindle KL Schnell RA Fernandez E amp Lefebvre PA (1989) Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase ndash J Cell Biol 109 2589ndash2601

Koutoulis A Pazour GJ Wilkerson CG Inaba K Sheng H Takada S amp Witman GB (1997) The Chlamydomonas reinhardtii ODA3 gene encodes a protein of the outer dynein arm docking complex ndash J Cell Biol 137 1069ndash1080

Kundeti VK Rajasekaran S Dinh H Vaughn M amp Thapar V (2010) Effi cient parallel and out of core algorithms for constructing large bi-directed de Bruijn graphs ndash BMC Bioinformatics 11 560

Landsberg JH (2002) The effects of harmful algal blooms on aquatic organisms ndash Fisheries Sci 10 113ndash390

Mali P Yang L Esvelt KM Aach J Guell M DiCarlo JE Norville JE amp Church GM (2013) RNA-guided human genome engineering via Cas9 ndash Science 339 823ndash826

Manuell AL amp Mayfi eld SP (2006) A bright future for Chlamydomonas ndash Genome Biol 7 327Mayfi eld SP amp Kindle KL (1990) Stable nuclear transformation of Chlamydomonas reinhardtii

by using a C reinhardtii gene as the selectable marker ndash Proc Nat Acad Sci USA 87 2087ndash2091

Merchant SS Prochnik SE Vallon O Harris EH Karpowicz SJ Witman GB Terry A Salamov A et al (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions ndash Science 318 245ndash250

Metzger P amp Largeau C (2005) Botryococcus braunii a rich source for hydrocarbons and related ether lipids ndash Appl Microbiol Biotechnol 66 486ndash496

Mock T amp Hipkin RH (2012)) Enhancer of cell division International Search Report Organiza-tion W I P Great Britain WO 2012107780 A1

Moellering ER amp Benning C (2010) RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii ndash Eukaryot Cell 9 97ndash106

eschweizerbart_xxx

24 Jianhua Liu

Niehaus TD Okada S Devarenne TP Watt DS Sviripa V amp Chappell J (2011) Identifi ca-tion of unique mechanisms for triterpene biosynthesis in Botryococcus braunii ndash Proc Nat Acad Sci USA 108 12260ndash12265

Palenik B Grimwood J Aerts A Rouze P Salamov A Putnam N Dupont C Jorgensen R et al (2007) The tiny eukaryote Ostreococcus provides genomic insights into the paradox of plankton speciation ndash Proc Nat Acad Sci USA 104 7705ndash7710

Pan YY Wang ST Chuang LT Chang YW amp Chen CN (2011) Isolation of thermo-tolerant and high lipid content green microalgae oil accumulation is predominantly controlled by photo-system effi ciency during stress treatments in Desmodesmus ndash Bioresour Technol 102 10510ndash10517

Persoone G amp Sorgeloos P (1975) Technological improvements for the cultivation of invertebrates as food for fi shes and crustaceans ndash Aquaculture 6 275ndash289

Polle JEW amp Qin S (2009) Development of genetics and molecular tool kits for species of the unicellular green alga Dunaliella (Chlorophyta) Enfi eld NH USA Sci Publ

Porath JB Gokham I Lers A Levy H amp Zamir A (1997) Developing a transformation system for Dunaliella ndash Phycologia 36 89

Prochnik SE Umen J Nedelcu AM Hallmann A Miller SM Nishii I Ferris P Kuo A et al (2010) Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri ndash Science 329 223ndash226

Rasala BA Lee PA Shen Z Briggs SP Mendez M amp Mayfi eld SP (2012) Robust expres-sion and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide ndash PLoS One 7(8) e43349

Rhode CJ (2012) Making fuel from algae identifying fact amid fi ction ndash In Gordon R amp Seck-bach J (eds) The science of algal fuel pp 177ndash192 Springer

Rismani-Yazdi H Haznedaroglu BZ Bibby K amp Peccia J (2011) Transcriptome sequencing and annotation of the microalgae Dunaliella tertiolecta pathway description and gene discovery for production of next-generation biofuels ndash BMC Genomics 12 148

Rismani-Yazdi H Haznedaroglu BZ Hsin C amp Peccia J (2012) Transcriptomic analysis of the oleaginous microalga Neochloris oleoabundans reveals metabolic insights into triacylglyceride accumulation ndash Biotechnol Biofuels 5 74

Ruan Y amp Wei CL (2010) Multiplex parallel pair-end-ditag sequencing approaches in system bi-ology ndash Wiley Interdiscip Rev Syst Biol Med 2 224ndash234

Shimogawara K Fujiwara S Grossman A amp Usuda H (1998) High-effi ciency transformation of Chlamydomonas reinhardtii by electroporation ndash Genetics 148 1821ndash1828

Sills DL Paramita V Franke MJ Johnson MC Akabas TM Greene CH amp Tester JW (2013) Quantitative uncertainty analysis of Life Cycle Assessment for algal biofuel production ndash Environ Sci Technol 47 687ndash694

Singer T amp Burke E (2003) High-throughput TAIL-PCR as a tool to identify DNA fl anking inser-tions ndash Methods Mol Biol 236 241ndash272

Sizova I Greiner A Awasthi M Kateriya S amp Hegemann P (2013) Nuclear gene targeting in Chlamydomonas using engineered zinc-fi nger nucleases ndashPlant J 73 873ndash882

Smith D (2012) Opinion Learning from Transcriptomes The ScientistSmith EF amp Lefebvre PA (1996) PF16 encodes a protein with armadillo repeats and localizes to

a single microtubule of the central apparatus in Chlamydomonas fl agella ndash J Cell Biol 132 359ndash370

Smith EF amp Lefebvre PA (1997) PF20 gene product contains WD repeats and localizes to the intermicrotubule bridges in Chlamydomonas fl agella ndash Mol Biol Cell 8 455ndash467

Sun D Zhu J Fang L Zhang X Chow Y amp Liu J (2013) De novo transcriptome profi ling uncovers a drastic downregulation of photosynthesis upon nitrogen deprivation in the nonmodel green alga Botryosphaerella sudeticus ndash BMC Genomics 14 715

eschweizerbart_xxx

Perspectives of green microalgal research for biofuels 25

Tam LW amp Lefebvre PA (1993) Cloning of fl agellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis ndash Genetics 135 375ndash384

Tan C Qin S Zhang Q Jiang P amp Zhao F (2005) Establishment of a micro-particle bombard-ment transformation system for Dunaliella salina ndash J Microbiol 43 361ndash365

Tang H Chen M Garcia ME Abunasser N Ng KY amp Salley SO (2011) Culture of micro-algae Chlorella minutissima for biodiesel feedstock production ndash Biotechnol Bioeng DOI 101002bit23160

Wake LV amp Hillen LW (1980) Study of a ldquobloomrdquo of the oil-rich alga Botryococcus braunii in the Darwin River Reservoir ndash Biotech Bioeng 22 1637ndash1656

Wang H Yang H Shivalila CS Dawlaty MM Cheng AW Zhang F amp Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPRCas-mediated genome engineering ndash Cell 153 910ndash918

Wang ZT Ullrich N Joo S Waffenschmidt S amp Goodenough U (2009) Algal lipid bodies stress induction purifi cation and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii ndash Eukaryot Cell 8 1856ndash1868

Weiss TL Chun HJ Okada S Vitha S Holzenburg A Laane J amp Devarenne TP (2010) Raman spectroscopy analysis of botryococcene hydrocarbons from the green microalga Botryo-coccus braunii ndash J Biol Chem 285 32458ndash32466

Wender PA Mitchell DJ Pattabiraman K Pelkey ET Steinman L amp Rothbard JB (2000) The design synthesis and evaluation of molecules that enable or enhance cellular uptake pep-toid molecular transporters ndash Proc Nat Acad Sci USA 97 13003ndash13008

Wijffels RH amp Barbosa MJ (2010) An outlook on microalgal biofuels ndash Science 329 796ndash799Worden AZ Lee JH Mock T Rouze P Simmons MP Aerts AL Allen AE Cuvelier ML

et al (2009) Green evolution and dynamic adaptations revealed by genomes of the marine pi-coeukaryotes Micromonas ndash Science 324 268ndash272

Work VH Radakovits R Jinkerson RE Meuser JE Elliott LG Vinyard DJ Laurens LM Dismukes GC et al (2010) Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains ndash Eukaryot Cell 9 1251ndash1261

Wu Z amp Shi X (2007) Optimization for high-density cultivation of heterotrophic Chlorella based on a hybrid neural network model ndash Lett Appl Microbiol 44 13ndash18

Wykoff DD Davies JP Melis A amp Grossman AR (1998) The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii ndash Plant Physiol 117 129ndash139

Xia L Ge H Zhou X Zhang D amp Hu C (2013) Photoautotrophic outdoor two-stage cultiva-tion for oleaginous microalgae Scenedesmus obtusus XJ-15 ndash Bioresour Technol 144 261ndash267

Zhang XH amp Chiang VL (1996) Single-stranded DNA ligation by T4 RNA ligase for PCR clon-ing of 5rsquo-noncoding fragments and coding sequence of a specifi c gene ndash Nucleic Acids Res 24 990ndash991

Zhao T Li G Mi S Li S Hannon GJ Wang XJ amp Qi Y (2007) A complex system of small RNAs in the unicellular green alga Chlamydomonas reinhardtii ndash Genes Dev 21 1190ndash1203

Zhao X amp Yu YT (2008) Targeted pre-mRNA modifi cation for gene silencing and regulation ndash Nat Methods 5 95ndash100

Zhou X Xia L Ge H Zhang D amp Hu C (2013) Feasibility of biodiesel production by micro-algae Chlorella sp (FACHB-1748) under outdoor conditions ndash Bioresour Technol 138 131ndash135

Manuscript received June 27 2013 accepted November 7 2013

eschweizerbart_xxx