Metabolic Engineering of Algae For Fourth Generation Biofuels Production

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Environmental Science
Cite this: Energy Environ. Sci., 2011, 4, 2451
www.rsc.org/ees REVIEW
Metabolic engineering of algae for fourth generation biofuels production
u,a Con Sheahanb and Pengcheng Fu*a
Jing L€
Received 25th October 2010, Accepted 14th March 2011
DOI: 10.1039/c0ee00593b
Published on 19 April 2011 on http://pubs.rsc.org | doi:10.1039/C0EE00593B
The ecological footprint and economic performance of the current suite of biofuel production methods
make them insufficient to displace fossil fuels and reduce their impact on the inventory of Green House
Downloaded by North Carolina State University on 09 December 2012
Gas (GHG) in the global atmosphere. Algae metabolic engineering forms the basis for 4th generation
biofuel production which can meet this need. The first generation biofuels are known to be made from
agricultural products such as corn or sugarcane. The second generation biofuels use all forms of (lingo)
cellulosic biomass. The third and fourth generation of biofuel production involves ‘‘algae-to-biofuels’’
technology: the former is basically processing of algae biomass for biofuel production, while the latter is
about metabolic engineering of algae for producing biofuels from oxygenic photosynthetic
microorganisms. Our review focuses on the research achievement of metabolic engineering of algae for
biofuel production. It is concluded that 4th generation biofuel production has introduced the ‘‘cell
factory’’ concept in this field, and shifted the research paradigm. There still exists several technical
bottlenecks in algae biofuel research and development, which can only be solved by the use of post-
genome tools on these photosynthetic organisms.
1. Introduction economy. As shown in Fig. 1, it is believed that there have been

four generations of biofuel production methods so far for the
Since the industrial revolution started in the UK about 250 years development of alternative energy. Most of the current biofuel
ago, the world’s economy has relied heavily on fossil fuels as an production is from the fermentation of sugar produced from
energy source. With rising fossil fuel prices and concerns about grains by conventional yeast strains, or on transesterification by
energy security, environmental pollution and climate change, the acid/alkali or enzyme based catalysts. It is the first generation of
quest for sustainable and renewable biofuel production has been biofuel production which is thought to have negative impacts on
gaining momentum over the past few years. As a result, renew- food security and controversial energy balance.1 Second gener-
able bioenergy and biofuels have drawn increasing attention in ation biofuels involve biological processing of (ligno)cellulosic
our society for use in a sustainable and environmentally friendly biomass to overcome the fuel vs. food dilemma.2 Both 3rd and
4th generation biofuels use photosynthetic microorganisms to
a
State Key Laboratory of Heavy Oil Processing, Unconventional Energy create renewable fuels: the former is basically processing of algae
Research Center, Faculty of Chemical Engineering, China University of biomass for biofuel production, while the latter is about meta-
Petroleum, Beijing, 18 Fuxue Road, Changping District, Beijing, 102249, bolic engineering of algae for producing biofuels from oxygenic
China. E-mail: pengcheng@cup.edu.cn; Fax: + 86-10-6074-3667; Tel: photosynthetic organisms.3 Algae are aquatic, photosynthetic
+86-10-9873-1283
b
Department of Manufacturing and Operations Engineering, Faculty of microorganisms that are abundant in marine environments, as
Science and Engineering, University of Limerick, Limerick, Ireland well as fresh water and brackish water all around the world. They
Broader context
The energy crisis and climate change have become the two main topics in all summit conferences since the beginning of this
millennium. It has been found that algae may be an ideal production system for renewable biofuels since these photosynthetic
organisms are easy to grow in fresh water, sea water or brackish water, and consume CO2 as their carbon source to convert solar
energy into chemical energy for cellular activities. With the aid of metabolic engineering, we are able to modify algae to produce
biofuels continuously while they are grown in ponds or closed photobioreactor systems. This approach possesses great potential in
providing sustainable and clean energy for our society to replace fossil fuels. Our review article concludes that the typical amount of
process steps required for biofuel production using 4th generation biofuel production methods is much less than the prior methods,
thereby avoiding large capital and operational expenditures.
This journal is ª The Royal Society of Chemistry 2011 Energy Environ. Sci., 2011, 4, 2451–2466 | 2451
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responsible for 50% of CO2 sequestration and 50% of O2

genesis in the world.5 Unless otherwise specified, for convenience
we will hereafter use the term ‘‘algae’’ to refer to the two major
categories of plant-like photosynthetic microorganisms; cyano-
bacteria (prokaryotes) and microalgae (eukaryotes). Either as
prokaryotes or as eukaryotes, algae have been isolated from fresh
and saltwater and soil. Thermophilic and halophilic variants
have also been isolated. Some of them live as symbionts in
sponges and plant roots. Nitrogen-fixing forms of cyanobacteria
act as natural fertilizers for rice and other crops. Some algae
secrete powerful toxins and terpenoid odorant compounds. They
are motile, and exhibit chemotaxis and phototaxis. A few species
of algae reportedly degrade to polynuclear aromatic hydrocar-
bons and other constituents of crude oil and refined petroleum

products.6 Others have potential applications in CO2 capture and
remediation.7 Astrobiologists are also interested in them as

a model life form that may have been able to exist before plants
developed on Earth, and perhaps in the early atmosphere on
Mars (http://amesevents.arc.nasa.gov/lunar/).
Algae metabolic engineering forms the basis for 4th generation
biofuel production. It uses recombinant DNA and other biological
Fig. 1 Four generations of biofuel production: from agricultural and bioengineering techniques for directed modification of cellular
products to algae. metabolism and properties through the introduction, deletion,
and/or modification of algal metabolic networks to create or
enhance biofuel production.8 This review article is not intended to
provide a thorough survey of the numerous metabolic modifica-
have been attracting renewed interest recently for world wide tion strategies for commercial applications of algae. Instead, we
algae-to-biofuel efforts due to soaring oil prices, and concerns have tried to illustrate algae-to-biofuel technology using metabolic
over environmental pollution and green house effects. engineering for biofuel production from photosynthetic microor-
Cyanobacteria are among the oldest forms of life on Earth, ganisms in the following five areas: (i) general algae gene trans-
appearing in the fossil record as much as 3.5 billion years ago.4 formation, (ii) gaseous biofuels, (iii) algal ethanol, (iv) algal
Cyanobacteria and microalgae are major players in the global butanol and high carbon alcohols, and (v) algal biodiesel.
carbon balance and oxygen generation as they are currently
2. Algae metabolic engineering

Dr Jing L€ u is currently an associate professor in the Faculty of 2.1 Cyanobacteria
Chemical Engineering, China University of Petroleum, Beijing.
She received her Ph.D. in the College of Biological Sciences from Cyanobacteria have become a research focus in the bioenergy
China Agriculture University in 2005. Her areas of research and biofuel fields. As the first application example, the larvicidal
interests include bioenergy and biofuels, genetic engineering, and gene of Bacillus sphaericus 1593M was transformed into the
algae and plant physiology.
Dr Pengcheng (Patrick) Fu is
Dr Con Sheahan is currently currently a professor in the
a senior lecturer in the Depart- Faculty of Chemical Engi-
ment of Manufacturing and neering, China University of
Operations Engineering, Petroleum, Beijing. He received
Faculty of Science and Engi- his BS and MS in Chemical
neering, University of Limerick, Engineering from Zhejiang
Ireland. He received his B.Eng. University, China, in 1982 and
in Production Engineering from 1988, respectively. He then
the University of Limerick in obtained his Ph.D. in Biochem-
1986. He then obtained his Ph. ical Engineering from the
D. in Integrated Systems Engi- University of Sydney, Australia,
neering from the University of in 1996. His areas of research
Limerick in 1996. His areas of Pengcheng Fu include bioenergy and biofuels,
Con Sheahan research interests include enter- metabolic engineering, bio-
prise performance modelling of process control, functional
bioenergy and biofuel supply genomics, systems biology and
chains. synthetic biology.
2452 | Energy Environ. Sci., 2011, 4, 2451–2466 This journal is ª The Royal Society of Chemistry 2011
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cyanobacterium Anacystis nidulans R2 in 1987.9 More recently, fusions to highly expressed endogenous proteins,145 or to highly
the genomic revolution has significantly advanced metabolic expressed recombinant proteins in the chloroplast.146
engineering for many photosynthetic organisms. As a milestone, Besides Chlamydomonas reinhardtii, Chlorella is another
Synechocystis sp. PCC 6803 (Synechocystis) has become the first unicellular green algae transformation system.96 Jarvis97 has
photosynthetic organism for which the genome was completely described the development of a transient expression system for
sequenced. The length of this circular genome is 3 573 471 bp.10 Chlorella ellipsoidea using a heterologous gene, or firefly lucif-
A total of 3317 protein coding genes was assigned (http:// erase. El-Sheekh98 has created a transgenic expression system of
genome.kazusa.or.jp/cyanobase/Synechocystis). Synechocystis is Chlorella kessleri by using the gene for b-glucuronidase (GUS).
a freshwater, non-filamentous, non-nitrogen fixing cyanobac- The stable transformants exhibited GUS activity. Hawkins101 has
teria capable of both photoautotrophic and heterotrophic developed the PEG method to use Chlorella (C. vulgaris C-27 and
growth. The genomic information, coupled with the biochem- C. sorokiniana) to express a recombinant heterologous protein
istry and physiological information available for Synechocystis consisting of an extracellular secretion signal sequence inserted
sp. PCC 6803, has made this strain one of the most popular between a promoter region and a gene for human growth
hormone (hGH). In 2002, Kim et al. 147 transformed the proto-
organisms for genetic and physiological studies of photosyn-

thesis. Synechocystis is also used as a model system for the study plasts of C. ellipsoidea with a vector containing the flounder
of oxygenic photosynthesis of higher plants since the size of its growth hormone gene (fGH), and stable FGH protein expression
genome is relatively small compared to any higher plant systems. was detected in the engineered microalgae.
Marine diatoms have been a new research frontier due to their
widespread existence on the Earth and their adaptability to
a varying environment, and their substantial biomass production
2.2 Eukaryotic microalgae
in water. Establishment of the diatom genome program and
The biotechnology of eukaryotic microalgae has entered into transgenic technology has laid a sound foundation for diatomic
a rapid developing phase in recent years. The availability of genetic manipulation.148 The recombinant diatoms have found
genomic information for eukaryotic microalgae has greatly applications in both fundamental studies and industrial
facilitated fundamental study and industrial applications. Table production.149,137 Using recombinant DNA technology, Duna-
111–95 is the list for microalgal genome sequences which have been hay132,149 has engineered two species of diatoms to insert chime-
completed or are to be completed. rical plasmid vectors containing a bacterial antibiotic resistance
Transgenic technology of eukaryotic microalgae for exoge- neomycin phosphotransferase II (nptII) gene into the diatoms
nous gene transformation and expression began in the 1980s. It Cyclotella cryptica and Navicula saprophila.
generally resulted in the random integration of exogenous genes
into the nuclear genome. Selection markers, promoters, reporter
2.3 Manipulation of microalgal metabolism
genes, transformation technology and other genetic tools or
methods have been successfully applied to several microalgae. The recombinant DNA technique for algae has proven to be
More than 30 different microalgal species have been successfully capable of creating constructs for both prokaryotes and
engineered by gene transformation into the cellular nucleus, eukaryotes which may replicate and possess novel functions. In
chloroplasts and mitochondria. Table 2 lists the eukaryotic addition to direct metabolic pathway modification, algal meta-
microalgae which have been investigated with gene trans- bolic engineering also involves targeted improvement of cellular
formation.96–139 activities by manipulation of enzymatic, transport, and regula-
Metabolic engineering research of eukaryotic microalgae was tory functions of the photosynthetic cells using other biological
mainly focused on unicellular green algae (i.e. Chlamydomonas and engineering approaches. Algae are subjected to a wide range
reinhardtii, and chlorella vulgaris) and marine diatoms. Chla- of environmental stresses, such as temperature elevation,
mydomonas reinhardtii is the most studied eukaryotic microalgae. nutrient depletion, or other adverse external factors in their
It is an ideal model organism for the understanding of various growth environment.150 Consequently, cells will respond with
fundamental mechanisms of biological processes, such as altered metabolic programming to adapt to the varying envi-
oxygenic photosynthesis, circadian rhythms, and flagella ronment. Environmental stress has been successfully used as
biogenesis. Recently, C. reinhardtii has also been studied as a means to manipulate microalgal metabolism in favor of lipid
a potential algal system for anaerobic biohydrogen produc- accumulation during the cell cultures. For instance, Liu et al.151
tion.140 The chloroplast DNA map of C. reinhardtii was estab- demonstrated that a high initial iron concentration in the
lished in 1978.141 Successful genetic transformation has also been medium has induced elevated lipid accumulation in marine strain
conducted in the C. reinhardtii chromosome, chloroplast142 and C. vulgaris. Illman et al.152 found that nitrogen deficiency resulted
mitochondrial genome.143 It is obvious that algae possess in an increase in the cellular lipid contents for all five investigated
a number of advantages over higher plant systems for the Chlorella strains. Among them, C. emersonii, C. minutissima and
production of recombinant proteins.144 In particular, Chlamy- C. vulgaris showed an increase in lipid content of 63%, 56% and
domonas has been demonstrated to be capable of producing 40% of their dry weight, respectively. Thomas et al.153 observed
recombinant proteins, even those complex mammalian thera- the effect of nitrogen stress on the algae lipid fraction for
peutic proteins and monoclonal antibodies are available at Botryococcus, Isochrysis and Dunaliella species. They found that
commercially viable levels with existing production platforms.144 nitrate deficiency could cause the protein content and the chlo-
Further research efforts have been undertaken to improve rophyll level to decrease while carbohydrate and lipids would
protein accumulation and stability by the expression of cleavable exhibit a species-specific change. Santos et al.154 have shown that
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Table 1 List for microalgal genome sequences which have been completed or are to be completed
Condition Phylum Species Strain GenBank Length (nt) Genes Proteins References
Complete (nuclear genome sequencing)

Chlorophyta
Chlamydomonas reinhardtii CC-503 cw92 mt+ ABCN00000000 105,192,443 14,354 14,412 11,12
Chlorella variabilis NC64A ADIC01000000 46,200,000 9,791 9,791 13
Micromonas pusilla CCMP1545 ACCP00000000 21,706,984 10,248 10,242 14
Micromonas sp. RCC299 CP001335- CP001574 20,900,00 10,128 10,128 14
Ostreococcus lucimarinus CCE9901 CP000581- CP000601 13,200,000 7,651 7,615 15
Ostreococcus tauri OTH95 CR954201- CR954220 12,560,000 8,166 8,166 16
Volvox carteri f. nagariensis Eve ACJH00000000 125,467,762 14,437 14,436 17
Ochrophyta
Phaeodactylum tricornutum CCAP1055/1 ABQD00000000 24,612,623 9,479 9,488 18
Thalassiosira pseudonana CCMP1335 AAFD00000000 29,453,142 10,747 10,660 18,19
Rhodophyta
Cyanidioschyzon merolae 10D AP006483(DDBJ) 16,728,945 5,331 5,017 20,21

Completed (mitochondrion genomes sequenced)
Chlorophyta
Chaetosphaeridium globosum M1311 AF494279 56,574 77 46 22
Chara vulgaris AY267353 67,737 76 46 23
Chlamydomonas eugametos CCAP11/6A AF008237 22,897 20 14 24
Chlamydomonas reinhardtii CC-503 cw92 mt+ U03843 15,758 25 8 12,25
Chlorokybus atmophyticus SAG 48.80 EF463011 201,763 89 58 26
Dunaliella salina CCAP19/18 GQ250045 28,331 21 9 27
Mesostigma viride NIES-296 AF353999 42,424 70 41 28
Oltmannsiellopsis viridis NIES 360 DQ365900 56,761 63 36 29
Prototheca wickerhamii UTEX 1553 U02970 55,328 63 36 30
Pseudendoclonium akinetum UTEX 1912 AY359242 95,880 94 72 31
Scenedesmus obliquus KS3/2 UTEX 393 X17375 42,781 53 20 32
Cryptophyta
Rhodomonas salina CCMP1319 AF288090 48,063 73 44 33
Haptophyta
Emiliania huxleyi 1516 AY342361 29,013 48 21 34
Hemiselmis andersenii CCMP644 EU651892 60,553 74 44 35
Ochrophyta (Phaeophyceae)
Chattonella marina KA11-m-1 AB546636 44,772 69 41 36
Desmarestia viridis CCAP1306/14 AY500367 39,049 68 39 37
Dictyota dichotoma AY500368 31,617 66 38 37
Fucus vesiculosus 1753 AY494079 36,392 67 38 37
Laminaria digitata CCAP1321 AJ344328 38,007 67 39 38
Ochromonas danica CCAP933 AF287134 41,035 75 44 39
Pylaiella littoralis Kjellam AJ277126 58,507 79 52 40
Saccharina angustata SANGU AP011498 37,605 66 38 41
Saccharina coriacea SCORI AP011499 37,500 66 38 42
Saccharina diabolica SDIAB AP011496 37,657 66 38 43
Saccharina japonica SJAPO AP011493 37,657 66 38 44
Saccharina longipedalis SLOPE AP011497 37,657 66 38 45
Saccharina ochotensis SOCHO AP011495 37,656 66 38 46
Saccharina religiosa SRELI AP011494 37,657 66 38 47
Synedra acus (Kutz.) Skabitsch GU002153 46,657 62 35 48
Thalassiosira pseudonana CCMP1335 DQ186202 43,827 61 35 19
Prasinophyta
Micromonas sp. RCC299 FJ859351 47,425 79 39 49
Nephroselmis olivacea NIES-484 AF110138 45,223 70 40 50
Ostreococcus tauri OTH95 CR954200 44,237 78 43 51
Pedinomonas minor CCAP1965 AF116775 25,137 23 11 50
Polytomella capuana SAG 63-5 EF645804 12,998 21 7 52
Pycnococcus provasolii CCMP1203 GQ497137 24,321 36 18 53
Rhodophyta
Cyanidioschyzon merolae 10D D89861 32,211 62 34 54
Chondrus crispus Nova Scotia Z47547 25,836 57 29 55
Gracilariopsis andersonii HQ586060 27,036 47 27 56
Gracilariophila oryzoides HQ586059 25,161 44 23 56
Plocamiocolax pulvinata HQ586061 25,894 46 24 56
Porphyra purpurea AF114794 36,753 57 31 57
Completed (chloroplast genomes sequenced)
Bacillariophyta
Phaeodactylum tricornutum CCAP1055/1 EF067920 117,369 170 132 58
Chlorophyta
Bryopsis hypnoides SAG 7.86 GQ892829 153,429 111 69 59
Chara vulgaris DQ229107 184,933 148 105 60
Chlamydomonas reinhardtii CC3269 BK000554 203,828 109 69 25
Chlorokybus atmophyticus SAG 48.80 DQ422812 152,254 157 114 61
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Table 1 (Contd. )
Chlorella vulgaris C-27 AB001684 150,613 210 174 62

Floydiella terrestris UTEX 1709 GU196268 521,168 105 74 63
Helicosporidium sp. Ex Simulium jonesii DQ398104 37,454 54 26 64
Leptosira terrestris UTEX 333 EF506945 195,081 119 88 65
Oedogonium cardiacum SAG 575-1b EU677193 196,547 138 99 66
Oltmannsiellopsis viridis NIES-360 DQ291132 151,933 127 93 67
Mesostigma viride NIES-296 AF166114 118,360 148 105 68
Parachlorella kessleri SAG 211-11g FJ968741 123,994 126 84 69
Pseudendoclonium akinetum UTEX 1912 AY835431 195,867 142 105 70
Scenedesmus obliquus UTEX 393 DQ396875 161,452 113 77 71
Staurastrum punctulatum SAG 679-1 AY958085 157,089 138 103 72
Stigeoclonium helveticum UTEX 441 DQ630521 223,902 110 79 73
Zygnema circumcarinatum SAG 698-1a AY958086 165,372 140 103 72
Cryptophyta
Cryptomonas paramecium CCAP977/2a GQ358203 77,717 115 82 74
Rhodomonas salina CCMP1319 EF508371 135,854 186 146 75

Guillardia theta CCMP2712 AF041468 121,524 183 147 76
Monomastix sp. OKE-I FJ493497 114,528 112 82 77
Dinophyta
Durinskia baltica CS-38 GU591327 116,470 167 129 78
Kryptoperidinium foliaceum CCMP1326 GU591328 140,426 177 139 78
Glaucophyta
Aureococcus anophagefferens CCMP 1984 GQ231541 89,599 137 105 79
Cyanophora paradoxa (cyanelle) Pringsheim strain LB 555 U30821 135,599 192 149 80
Haptophyta
Emiliania huxleyi 1516 AY741371 105,309 154 119 34,81
Ochrophyta
Ectocarpus siliculosus Ec32 CCAP1310/4 FP102296 139,954 185 148 82
Heterosigma akashiwo CCMP452 NIES-293 EU168190 159,370 198 156 83
Odontella sinensis CCMP1762 Z67753 119,704 175 140 84
Thalassiosira pseudonana CCMP1335 EF067921 128,814 180 141 58
Vaucheria litorea CCMP2940 EU912438 115,341 172 139 85
Prasinophyta
Ostreococcus tauri OTH95 CR954199 71,666 94 61 51
Pycnococcus provasolii CCMP1203 FJ493498 80,211 101 68 77
Pyramimonas parkeae CCMP726 FJ493499 101,605 130 94 77
Micromonas pusilla CCMP1545 FJ858269 41,811 40 27 14
Micromonas sp. RCC299 FJ858267 72,585 32 57 86
Nephroselmis olivacea NIES-484 AF137379 200,799 200 155 87
Rhodophyta
Cyanidioschyzon merolae 10D AB002583 149,987 243 207 54
Cyanidium caldarium RK1 AF022186 164,921 230 197 88
Gracilaria tenuistipitata Liui AY673996 183,883 238 203 89
Porphyra purpurea Avonport U38804 191,028 253 209 90
Porphyra yezoensis TU-1 AP006715 191,952 264 209 91
Streptophyta
Chaetosphaeridium globosum M1311 AF494278 131,183 141 98 22
Chlorarachniophyceae
Bigelowiella natans CCMP2755 DQ851108 69,166 98 61 92
Euglenozoa
Euglena longa AJ294725 73,345 84 46 93
Euglena gracilis Pringsheim strain Z X70810 143,171 115 67 94
Ongoing (microalgal genome sequencing projects)
Aureococcus anophageferrens CCMP1984 95
Bigelowiella natans CCMP2755 95
Botryococcus braunii 95
Chaetosphaeridium globosum SAG 26.98 95
Chattonella subsalsa CCM 217 95
Chlorella vulgaris C-169 95
Coccomyxa sp. C-169 95
Cyclotella meneghiniana 95
Dunaliella salina CCAP19/18 95
Emiliania huxleyi 1516 95
Fragilariopsis cylindrus CCMP1102 95
Galdieria sulfuraria 95
Guillardia theta CCMP2712 95
Micromonas pusilla 95
Nannochloropsis oculata CCMP525 95
Ostreococcus sp. RCC809 95
Phaeocystis antarctica 95
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Table 1 (Contd. )
Pinguiococcus pyrenoidosus CCMP2188 95

Porphyra purpurea 95
Pseudo-nitzschia Multiseries CLN-47 95
Tribonema aequale CCMP1275 95
Volvox carteri 95
Table 2 Eukaryotic microalgae which have been investigated with gene transformation
Phylum Species Strain Organelle Gene Reference

Chlorophyta
Chlorella ellipsoidea Nuclear NP-1 96
Chlorella ellipsoidea CCAP211/1a Protoplast Luc 97

Chlorella kessleri 211-11h Nuclear Gus 98
Chlorella vulgaris Nuclear Hpt, gus 99
Chlorella sorokiniana Nuclear NR 100
Chlorella sorokiniana Chlorella vulgaris C-27 22521 Nuclear HGH 101
Chlorella saccharophila C-211-la Protoplast Gus 102
Chlamydomonas reinhardtii (Nit1-305) Nuclear NR 103
Chlamydomonas reinhardtii 137c (mt+) Chloroplast GFPct, GFPncb 104
Chlamydomonas reinhardtii Cw15 arg Nuclear Crluc 105
Chlamydomonas reinhardtii Cw15 argA Nuclear Cgfp 106
mt
Chlamydomonas reinhardtii Cw-15 Nuclear Nit 107
Chlamydomonas reinhardtii Nitl-305 Nuclear Nit 108
Chlamydomonas reinhardtii CC-124, CC- Nuclear UidA, gfp, hpt 109
125
Chlamydomonas reinhardtii J3 (mt) Nuclear COX90, PSY, DCL1 110
Chlamydomonas reinhardtii CC3395, Nuclear ARG7 111
CC425
Chlamydomonas reinhardtii CC-425 Nuclear MAA7, RBCS1/2 112
Chlamydomonas reinhardtii CW15-302, Nuclear PHOT 113
UV4
Dunaliella viridis Nuclear DvNIA1 112
Dunaliella salina Nuclear Ble 115
Dunaliella salina Nuclear Gus, bar 116
Haematococcus pluvialis Nuclear LacZ 117
Nannochloropsis oculata Plasmid Ypgh 118
Ulva lactuca Protoplast Gus 119
Volvox carteri HB11A Nuclear NitA 120
Rhodophyta
Porphyra yezoensis Nuclear Gus, gfp, RPB1, GAPDH 121
Gracilaria changii Nuclear LacZ 122
Porphyra miniata Protoplast Gus 123
Porphyridium sp Chloroplast AHAS (W492S) 124
Cyanidioschyzon merolae 10D Nuclear URA5.3 125
Phaeophyta
Laminaria japonica F003, M007 Sporophyte LacZ 126
Laminaria japonica Nuclear UidA, cat. lacZ 127
Undaria pinnatifida Nuclear LacZ 128
Bacillariophyceae
Phaeodactylum tricornutum Nuclear Sh ble, cat 129
Phaeodactylum tricornutum Bohlin Plastid Gfp 130
Phaeodactylum tricornutum Bohlin Nuclear Gus, dph1, cpf1 131
CCMP632
Cyclotella cryptica Navicula saprophila T13L NAVIC1 Nuclear NptII 132
Phaeodactylum tricornutum Bohlin 646 Chloroplast GtPGK, PtFBAC1, PtOEE1, PtFBAC, PtHLIP2, 133
PtFBPC4, PtFSA
Thalassiosira pseudonana CCMP1335 Nuclear Fcp, ble, nat1, NR 134
Cylindrotheca fusiformis Nuclear CfNR 135
Phaeodactylum tricornutum Nuclear Nat, sat-1, nptII uid, gfp 136
Phaeodactylum tricornutum Nuclear Glut1 137
Euglenids
Euglena gracilis Chloroplast AadA 138
Dinoflagellates
Amphidinium sp. Symbiodinium Nuclear NptII, hpt, gus 139
microadriaticum
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the lipid content in Spirulina increased by approximately three 633 genes, 704 metabolites and 831 metabolic reactions for the
times with the decrease of both nitrogen concentration and study of optimal Synechocystis growth, network capacity and
temperature. functions. The Synechocystis model was used for in silico
Improvement of solar conversion efficiency is of critical predictions of heterotrophic, photoautotrophic and mixotrophic
importance for the development of algae-to-biofuel technologies. growth conditions, and of the insertion of an ethanol fermenta-
Through their billion years of evolution, microalgae have tion pathway. Simulation results were also compared with actual
developed effective light-harvesting antennae complexes (LHCs) metabolic measurements which showed satisfactory agreement.
to capture light energy. Under strong light, their photosystems I More recently, two more papers on Synechocystis metabolic
and II can be overwhelmed by high light intensity which leads to models for photoautotrophic growth have been established in the
photoinhibition.155 In order to manipulate microalgal photo- literature.169,170
synthesis and metabolism, the Melis lab has worked on Chla- The metabolic network for green alga, C. reinhardtii, was
mydomonas cells with a truncated antenna size to create a C. reconstructed,171 accounting for the intracellular localization of
reinhardtii DNA insertional mutant, tla1.156 It was concluded enzymes to three compartments (mitochondria, chloroplastic
that fully pigmented cells would over-absorb the light and and cytosol). The C. reinhardtii model consists of 484 metabolic
wastefully dissipate heat which might reduce photosynthetic reactions and 458 intracellular metabolites. FBA was used to
efficiency, and that truncated Chl antenna cells permit greater estimate intracellular metabolic flux distributions under auto-
transmittance of light and a better overall solar utilization by the trophic, heterotrophic and mixotrophic growth conditions. The
microalgal cell cultures. Another group of researchers have used modeling efforts also provide a comprehensive method for
RNA interference technology to down-regulate the expression of annotation of genome databases.
LHC proteins in C. reinhardtii to reduce photodamage and
increase light penetration in microalgal cell growth.157 The
resulting LHC mutant was seen to show more efficient conver- 3. Case studies
sion of solar energy to biomass.
3.1 Gaseous biofuels
Oxygenic photosynthesis of algae can be utilized in renewable

2.4 Reconstruction and analysis of genome-scale metabolic
hydrogen production from solar energy and water.172 As
networks
a fermentation end product, H2 may be produced by a variety of
Algae metabolic networks are highly complex nonlinear reaction algal strains from an endogenous bidirectional hydrogenase;
webs which are tightly coordinated to carry out a large number from a native nitrogenase; or from an exogenous hydrogenase.173
of biochemical reactions in an organized manner, to meet the Genetic engineering plays an increasingly important role in
physiological demands of photosynthetic microorganisms and to eukaryotic microalgae modification for algal H2 production.
adapt to the cellular environment. Studies of bacterial metabolic Among numerous examples of applications, Kruse et al.174 in
networks have been undertaken for more than three decades.158 2005 have shown that improved hydrogen production in the
Models of intricate metabolic networks can be developed to green alga Chlamydomonas reinhardtii can be achieved by
make predictions or to explain the properties of biological modifying the strain’s respiratory metabolism. Doebbe et al.,175
systems. Simulation models in various forms have proved to be have reported that it could enhance bio-hydrogen production
powerful, flexible and efficient tools for the evaluation of system through heterologously expressed HUP1 (hexose uptake protein)
dynamics, for the understanding of structure and functions of in Chlamydomonas Reinhardtii, in 2007. Recombinant DNA
metabolic networks, and for the selection, modification, config- technology has been applied to algae to increase phototrophic H2
uration and capacity assessment of a living system. production, and numerous research articles have been published
There have been several modeling attempts to increase our in the literature on this topic. A detailed discussion is beyond the
biological knowledge of the in vitro behavior of metabolic scope of this paper. Interested researchers can read the survey
networks, but the constraint-based modeling approach using flux articles listed under Ref. 7 and 176 for more information.
balance analysis and linear programming159,160 is the only Another potential gaseous biofuel is isoprene (2-methyl-1,3-
methodology at present by which genome-scale models have butadiene), a volatile hydrocarbon that is insoluble in water. It
been constructed.161 This constraints-based genome-scale model can thus be separated as a gas in a convenient way. Traditionally
takes into account the largest metabolic details in terms of isoprene has been produced mainly through isopentane dehy-
numbers of genes and reactions. This kind of in silico metabolic drogenation, isobutylene carbonization and isoamylene dehy-
model has been proven to be effective in systems biology analysis drogenation. The most common route is extraction from the C5
of phenomic data,162–164 qualitative transcriptomic data,165 and stream as a cracking by-product of ethylene production.177 More
gene knockout data.166,167 The results thus far have shown than 95% of isoprene is used in the generation of industrial
a surprising degree of correlation between the predictions of polymers; polyisoprene, also known as isoprene rubber (IR)
genome-scale models and independently obtained experimental accounts for about 60% of the world’s rubber production and is
data.162,166 used primarily as a component of premium tyres, adhesive
Several genome-scale metabolic networks of cyanobacteria sealants and other rubber products.178 Bioisoprene can then be
and algae have appeared in the past couple years and are converted into jet fuel, diesel, polymers or synthetic rubber
important for metabolic engineering efforts in algae-to-biofuel through additional chemical processes. Plants are known to
research. For example, Fu (2009)168 reconstructed a genome- generate isoprene from CO2 via photosynthetic carbon fixation
scale Synechocystis PCC 6803 metabolic network, which includes and the mevalonic acid (mevalonate) pathway.179 In plants, the
View Article Online
last step in the biosynthesis of isoprene, cleavage of pyrophos- synthase in the cyanobacterium Anabeana 7120, wherein the
phate from isopentyl diphopshate (IPP) and dimethyl allyl gene lspS is maintained as a plasmid, has already been demon-
diphosphate (DMAPP) is carried out by a single enzyme, strated. Recently, Lindberg et al.184 have engineered a platform
isoprene synthase. for bioisoprene production using Synechocystis PCC 6803. They
There is precedence for the cloning of plant isoprene genes into have applied synthetic biology to the synthesis of the native
bacteria. For example, a cDNA clone for isoprene synthase has kudzu (kIspS) and Synechocystis codon-optimized kudzu IspS
been cloned from poplar.180 Expression of this foreign gene, lspS, (SkIspS) genes using the nucleotide sequence of the P. montana
in E. coli, and production of isoprene from the recombinant (kudzu) isoprene synthase gene (IspS)185 without its predicted
organism, has also been demonstrated.180 The class of enzymes to chloroplast transit peptide. These newly designed DNA
which isoprene synthase belongs, terpene cyclases, has been sequences were synthesized to encode isoprene synthase and
relatively well-studied. The 3D structures of the homologs 5-epi- catalyze the biochemical conversion of DMAPP to isoprene. The
aristolochene synthase181 and bornyl diphosphate synthase182 genes were transformed into Synechocystis PCC 6803 under the
have been determined. control of a light driven promoter psbAII. The over-expression of
As illustrated in Fig. 2, cyanobacteria are known to lack a gene ispS protein with high lighting intensity resulted in the accumu-
for isoprene synthase but can synthesize the penultimate isoprene lation of isoprene at a rate of 50 mg day1 per g dry cell mass.184
precursors (IPP and DMAPP) via the 2-C-methyl-D-erythritol-4- The metabolic flux relationship was analyzed to investigate the
phosphate (MEP) pathway rather than the mevalonic acid intracellular pathway utilization. The authors have thus found
pathway.183 Like plants, the carbon source for IPP and DMAPP that photosynthetic carbon partitioning among the sugar,
in cyanobacteria is obtained through the photosynthetic fixation terpenoid and fatty acid biosynthetic pathways needs to be
of carbon dioxide. Hence, a cyanobacterium metabolically reprogrammed in order to increase the carbon flow toward the
engineered to incorporate an expressible isoprene synthase gene isoprene synthesis via the terpenoid pathway. This work has
could generate isoprene directly from CO2 and water in the shown that green isoprene can be produced by photosynthetic
presence of sunlight. Cloning and expression of active isoprene cyanobacteria, through heterologous expression of the gene
encoding for the isoprene synthase (IspS), in a reaction of the
MEP pathway, driven by solar energy.184
3.2 Algal ethanol

Biologically produced ethanol as a fuel is an attractive renewable
energy concept. It has recently surged to the forefront of biofuel
technology. Most of the current ethanol production is from
biomass fermentation of agricultural crops and residues. The
baker’s yeast Saccharomyces cerevisiae and facultative bacterium
Zymomonas mobilis are the two well known ethanologenic
microbes for industrial alcohol production. The shortcomings in
using Z. mobilis for microbial ethanol production include: (i) its
inability to convert complex carbohydrate polymers like cellu-
lose, hemicellulose, and starch to ethanol; (ii) its byproducts,
such as sorbitol, acetoin, glycerol, and acetic acid; (iii) formation
of extracellular levan polymer.
Construction of the ethanol fermentation pathway is relatively
straightforward. Both pdc and adhII genes from Z. mobilis were
first transformed into E. coli for the expression of pyruvate
decarboxylase (PDC, EC: 4.1.1.1) and alcohol dehydrogenase II
(ADH, EC: 1.1.1.1) under the control of the lac operon.186–188
The idea of producing bioethanol in cyanobacteria through
Z. mobilis pdc/adhII gene transformation was first proposed by
Deng and Coleman.189 Their work demonstrated that ethanol
could be produced from CO2 fixation via photoautotrophic
metabolism. In their experiment, the Cyanobacterium Synecho-
coccus sp. strain PCC 7942 was transformed with pCB4-based
shuttle vectors bearing the Z. mobilis pdc/adh gene cassette, first
described by Ingram et al. in 1987.186 The two genes were
expressed under the control of the rbcLS operon promoter, both
alone and in combination with the E. coli lac promoter. Here the
rbcLS operon encodes the ribulose-1,5-bisphosphate carboxy-
lase/oxygenase large and small subunits. It is an endogenous
promoter (from Synechococcus PCC 7942) with the temperature
Fig. 2 The isoprene production pathway. inducible CI-PL promoter. The results showed that the
View Article Online
recombinant cyanobacteria would produce ethanol with the yield protein) and the lowest Km (0.24 mM). Similarly, the adh gene is
in an order of magnitude of 54 nmol OD730 unit1 litre1 day1. commonly found in many organisms. In 1988 Youngleson et al.
In the same study, ethanol concentrations of approximately 5 cloned an adhII gene into a recombinant plasmid, pCADH100,
mM were reached after 4 weeks of growth. A later patent from Clostridium acetobutylicum.192
describes yields of 1.7 mmol of ethanol per mg of chlorophyll per
hour with the CI-PL temperature inducible promoter (US Patent
3.3 Algal butanol and four carbon alcohols
No. 6699696, 5/21/2001).
More recently, Dexter and Fu (2009)190 have reported their Butanol is one of the four carbon alcohols that may be used as
metabolic engineering work on Synechocystis sp. PCC 6803 that a liquid biofuel. Compared to ethanol, butanol possesses a heat
can photoautotrophically convert CO2 to bioethanol (Fig. 3). content per weight similar to that of gasoline. It also has lower
Transformation was performed using a double homologous vapor pressure, and is hydrophobic.193 Therefore butanol may
recombination system to integrate the pyruvate decarboxylase become a potential transportation fuel to supplement gasoline.
(pdc) and alcohol dehydrogenase II (adh) genes from obligately Among the anaerobic microbes that are capable of butanol
ethanol producing Zymomonas mobilis into the Synechocystis production, Clostridium acetobylicum is a well known bacterium
chromosome under the control of the strong, light driven psbAII for sugar fermentation to produce butanol.194 The enzymes
promoter. The engineered Synechocystis has shown an average participating in the formation of butanol have been well char-
yield of 5.2 mmol OD730 unit1 liter1 day1 with no required acterized and the regulating genes been cloned.195 Genome
antibiotic/selective agent during the photobioreactor cultures. information regarding C. acetobylicum is also available. A rele-
Comparison of the mass balances for ethanol production by both vant genome-scale model using flux balance analysis and a linear
the yeast Saccharomyces cerevisiae and the cyanobacterium program can also be used to understand the structure and
Synechocystis PCC 6803 indicates that the theoretical yield of function of the C. acetobylicum metabolic network.196
ethanol from glucose for S. cerevisiae is 0.51 g ethanol/g glucose, After successful metabolic engineering of E. coli for the
while the theoretical yield of ethanol from CO2 for Synechocystis production of butanol and high carbon alcohols, the Liao group
is 0.696 g ethanol/g CO2. has moved on to metabolic engineering of the photosynthetic
It is obvious that pdc and adhII genes play an important role in organism, Synechococcus elongatus PCC7942 to produce
the construction of bioethanol-producing cyanobacteria by butanol, isobutyraldehyde and isobutanol directly from CO2.
metabolic engineering. Therefore, algal ethanol yield and effi- The genetically engineered S. elongatus was active for 8 days and
ciency may be improved by transformation and expression of produced isobutyraldehyde at a higher rate than those reported
these two genes from other organisms. Raj et al.191 have for ethanol, hydrogen or lipid production by cyanobacteria or
compared the biochemical and kinetic properties of the pyruvate algae.197 Fig. 4 shows the butanol production pathway created
decarboxylase enzyme from Zymobacter palmae with purified for the bioconversion of CO2 to butanol by photosynthetic
pdc from three other bacteria. It was found that the Z. palmae organisms. The kivd gene for ketoacid decarboxylase from
enzyme exhibited the highest specific activity (130U per mg of Lactococcus lactis was transformed into S. elongatus by an
expression cassette under the control of the isopropyl-b-D-thio-
galactoside (IPTG) inducible promoter Ptrc.197
3.4 Algal biodiesel

Algal biodiesel is a hot topic in biofuel production since it performs
similar to petroleum diesel whilst being beneficial to the environ-
ment by substantial reduction of unburned hydrocarbons (carbon
monoxide) in comparison to emissions from diesel fuel. Biodiesel
can be used in existing diesel engines with little impact on operating
performance. In addition, it has a similar gross heat value (126 200
BTU gal1) to that of diesel fuel (138 700 BTU gal1).198
Fatty acids (FA) are precursors for biodiesel production. The
FA synthesis involves the conversion of acetyl CoA to malonyl
CoA, catalyzed by acetyl CoA carboxylase (ACCase). The
associated pathway produces 16- to 20-carbon fatty acids for the
synthesis of cellular and organelle membranes as well as for the
synthesis of storage lipids, mainly triacylglycerols (TAGs). Some
oligeanous microalgae will accumulate TAG as high as 30–60%
of dry cell weight, which is at least an order of magnitude higher
than terrestrial crop plants.199 The major TAG synthesis
pathway for lipid accumulation in algae occurs primarily in the
chloroplast.
In order to increase the biodiesel yield, oligeanous algae, yeast,
and even bacteria have been screened for lipid content. It was
Fig. 3 The ethanol production pathway. found that the TAG contents for some microalgal species can
View Article Online
reach 60–80% of their total biomass.200 However, several tech- 4. Conclusion

nical barriers need to be overcome for the commercialization of
these species. These include low growth rates, difficulties in large Algae biotechnology has recently attracted increasing interest
scale culture and utilization of environmental stress for lipid due to its substantial potential in solving bioenergy problems and
synthesis. Stress control strategies are usually developed by emission reduction. Metabolic engineering of algae has emerged
controlling the nutritional or cultivation conditions (i.e., to open up a new vista for rapid advancement of photosynthetic
temperature, pH, nitrogen, phosphate concentrations, etc.) to organisms, in particular aquatic ones, in the bioenergy and bio-
reprogram microalgal cells from normal growth to high lipid fuel, environmental protection and emission reduction areas.
accumulation for their adaptation to the changing Based on the survey above, we can draw conclusions on the
environment.201 following three aspects:
Recombinant DNA technology to improve biodiesel produc-
tion has recently been approached. Isolation and characteriza-
tion of an acetyl-CoA carboxylase (ACCase) gene from 4.1 The 4th generation of biofuels and its counterparts
a photosynthetic organism, C. cryptica was conducted in 1993.202
The ACCase gene was then successfully transformed into the The main difference between 4th generation biofuel production
diatoms C. cryptica and Navicula saprophila. However, the lipid and the previous approaches is that only the former one applies
content of the microalgae was not increased significantly.149 the ‘‘cell factory’’ concept for the production of gaseous and
Nevertheless, Beer et al. have designed strategies to engineer fatty liquid biofuels. The photosynthetic organisms are thus driven by
acid (FA) biosynthesis in plants as follows: Firstly, to over- solar energy from the sun for continuous production of biofuels
express FA biosynthetic enzymes; secondly, to increase the using CO2 as the raw material. The common feature of 4th
availability of precursor molecules, such as acetyl-CoA; thirdly, generation biofuel production methods is that they will secrete
to down-regulate FA catabolism by inhibiting b-oxidation, or the end products out of the cells, which will avoid the costly
lipase hydrolysis; fourthly, to introduce the desaturases to alter fermentation and/or processing of biomass feedstock applied in
saturation profiles; and finally, to optimize FA chain length with current biofuel production.
thioesterases.176 It should be noted that the most critical benefit in 4th gener-
ation biofuel production is the minimization of the number of
process steps between the sun providing energy and the trans-
formation of this energy into a fungible biofuel and capturing
CO2 prior to its emission into the environment. This benefit will
need to be matched to an appropriate biofuel type so that it
exploits the existing fossil fuel distribution infrastructure. It is
evident from Fig. 5 that the typical process steps required for
biofuel production using 4th generation methods are much less
than with the prior methods, thereby avoiding large capital and
operational expenditures.
4.2 Advantages and disadvantages of biofuel production by

photosynthetic organisms
There exists both advantages and disadvantages of 4th genera-
tion biofuel production. For example, isoprene represents
a gaseous biofuel which will be easily separated from the
production broth. The disadvantages include that bioisoprene
production requires more expensive closed photobioreactor
systems to prevent leaking of the product, and that isoprene
needs to be separated from the exhaust gases of the photo-
bioreactors. For bioethanol production, its metabolic engi-
neering is relatively straightforward. However, ethanol possesses
high water content and relatively low heat content in comparison
to gasoline, which means ethanol is not an ideal replacement for
fossil fuels. Biobutanol is hydrophobic and the gasoline-butanol
blends do not separate in the presence of water. There is no need
to modify gasoline engines. Nevertheless, its energy content is
similar to that of gasoline. On the other hand, butanol has a legal
impediment from the U.S. federal government so that butanol is
not recognized as a biofuel yet. Biodiesel possesses the highest
heat content of other algae biofuels. Biodiesel is less suitable for
Fig. 4 The butanol production pathway. use in low temperatures than petrodiesel.
View Article Online
Fig. 5 Comparison of the typical bioprocess steps required for four generations of biofuels production.
4.3 Bottlenecks for large scale production AtoB Acetyl-CoAacetyltransferase

While the basic concept of using algae as an alternative and Bcd Butyryl-CoAdehydrogenase
renewable source of biofuels has been explored over the past BUTCOA Butyryl-CoA
several decades, the development of 4th generation biofuel BUTDEH Butyraldehyde
production is still in its infancy. While the economic feasibility of CCOA Crotonyl-CoA
4th generation fuels appears superior in the longer term, there are CDP-ME Diphosphocytidylylmethylerythritol
still technical risks that will have to be overcome in the provision CDP-MEP CDP-ME2-phosphate
of supporting infrastructure. Even with a high performing engi- Crt Crotonase
neered microalgae the balance of the plant design and associated DMAPP Dimethylallyldiphosphate
knowledgebase does not exist for 4th generation biofuels to the DXP Deoxyxylulose5-phosphate
extent that it does for other biofuel production methods. The Dxr DXP reductoisomerase
most salient issues are the provision of cost effective photo- Dxs DXPsynthase
bioreactors and biofuel separation technologies with an excellent Etf Electrontransfer flavoprotein
energy balance. GcpE (E)-4-hydroxy-3-methylbut-2-enyl-
The main barriers are: 1) our lack of understanding of algal diphosphate synthase
growth, metabolism and biofuels production. 2) The accumula- Hbd 3-Hydroxybutyryl-CoAdehydrogenase
tion of biofuel metabolites for some algae may cause cytotoxicity HMBPP Hydroxymethylbutenyldiphosphate
to the host cells. To solve this problem, cellular tolerance to the Ipi IPPisomerase
accumulated biofuels needs to be developed either by metabolic IPP Isopentenyldiphosphate
engineering or by directed evolution. 3) Over-expression of IspD CDP-MEsynthase
exogenous enzymes in algae may disturb the native metabolism IspE CDP-MEkinase
by competing for the precursors necessary for cellular activities IspF ME-cPP synthase
and maintenance. Balancing the altered metabolic network is IspG HMBPPsynthase
needed to eliminate the bottleneck in the biosynthetic pathway IspH HMBPPreductase
that diminishes biofuel production. 4) Since the enzymes regu- IspS Isoprenesynthase
lated by foreign genes for 4th generation biofuel production are ME-CPP Methylerythritol2: 4-cyclodiphosphate
not evolved to fully function in oxygenic photosynthetic organ- MEP Methylery-thritol4-phosphate
isms, their performance would not be optimal. As a result, it pdc Pyruvate decarboxylase
should be kept in mind that no matter what novel fuel molecules PBR Photobioreactors
could be made from algae, it is impossible to make an impact on PP Pentose phosphate
the fuel market until the technology is economically feasible. PYR Pyruvate
Abbreviations
Acknowledgements
3HBCOA 3-Hydroxybutyryl-CoA This work is supported by the National Basic Research Program
AACoA Acetoacetyl CoA of China (973 program) under grant No. 2011CB200902. This
ACAL Acetaldehyde work is also partially funded by PetroChina Company Ltd. in
ACCoA Acetyl-CoA China. The authors are grateful to the three anonymous
adh Alcohol dehydrogenase reviewers for their critical and constructive comments which
AdhE2 Aldehyde/alcoholdehydrogenase have improved this manuscript.
View Article Online
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Energy & Dynamic Article Links < C

1. Introduction economy. As shown in Fig. 1, it is believed that there have been

responsible for 50% of CO2 sequestration and 50% of O2

bons and other constituents of crude oil and refined petroleum

remediation.7 Astrobiologists are also interested in them as

2. Algae metabolic engineering

organisms for genetic and physiological studies of photosyn-

Complete (nuclear genome sequencing)

Cyanidioschyzon merolae 10D AP006483(DDBJ) 16,728,945 5,331 5,017 20,21

Chlorella vulgaris C-27 AB001684 150,613 210 174 62

Rhodomonas salina CCMP1319 EF508371 135,854 186 146 75

Pinguiococcus pyrenoidosus CCMP2188 95

Phylum Species Strain Organelle Gene Reference

Chlorella ellipsoidea CCAP211/1a Protoplast Luc 97

Oxygenic photosynthesis of algae can be utilized in renewable

3.2 Algal ethanol

3.4 Algal biodiesel

reach 60–80% of their total biomass.200 However, several tech- 4. Conclusion

4.2 Advantages and disadvantages of biofuel production by

4.3 Bottlenecks for large scale production AtoB Acetyl-CoAacetyltransferase

References 24 E. M. Denovan-Wright, A. M. Nedelcu and R. W. Lee, Complete

Netherlands. 29 J. F. Pombert, P. Beauchamp, C. Otis and C. Lemieux, et al., The

for mitochondrial transformation, MGG, Mol. Gen. Genet., 1993, 5533.

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