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Diatoms: a fossil fuel of the future
Opinion
Diatoms: a fossil fuel of the future Orly Levitan1, Jorge Dinamarca1, Gal Hochman2, and Paul G. Falkowski1,3 1
Environmental Biophysics and Molecular Ecology Program, Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ 08901, USA 2 Department of Agriculture, Food & Resource Economics, Rutgers University, New Brunswick, NJ 08901, USA 3 Department of Earth and Planetary Sciences, Rutgers University, Piscataway, NJ 0885, USA
Long-term global climate change, caused by burning petroleum and other fossil fuels, has motivated an urgent need to develop renewable, carbon-neutral, economically viable alternatives to displace petroleum using existing infrastructure. Algal feedstocks are promising candidate replacements as a ‘drop-in’ fuel. Here, we focus on a specific algal taxon, diatoms, to become the fossil fuel of the future. We summarize past attempts to obtain suitable diatom strains, propose future directions for their genetic manipulation, and offer biotechnological pathways to improve yield. We calculate that the yields obtained by using diatoms as a production platform are theoretically sufficient to satisfy the total oil consumption of the US, using between 3 and 5% of its land area. The need for carbon-neutral fuels The first major oil well, drilled in 1859 by Edwin Drake, supplied cheap fuel for kerosene lamps, and led to a dramatic reduction in the demand for whale blubber. Although the use of kerosene as a fuel for lighting can be claimed as saving whales from being hunted to extinction, there were other unintended consequences to follow. The subsequent invention of internal combustion engines provided a huge demand for gasoline, which previously had been a worthless byproduct of kerosene distillation. By the early decades of the 20th century, the oil industry had become the engine of economic growth in industrializing nations. A legacy of Drake’s oil well is that over 150 years later, 96% of all transportation processes in the world is still based on petroleum [1]. The proven global reserves are projected to be able to meet the predicted demand for several decades (http://energy.gov/fe/ services/petroleum-reserves). However, an unintended consequence of the rapid combustion of fossil fuels is the rise in greenhouse gas emissions. Since the beginning of the Industrial Revolution, >350 billion metric tons of carbon have been emitted into the atmosphere, with a commitment rise in atmospheric CO2 of 43% over the past 150 years (http:// petrolog.typepad.com/climate_change/2010/01/cumulativeemissions-of-co2.html, http://www.esrl.noaa.gov/gmd/ccgg/ Corresponding authors: Levitan, O. ([email protected]); Dinamarca, J. ([email protected]). Keywords: biofuel; diatoms; lipids; biomass; productivity; Energy Return (On) Investment. 0167-7799/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.01.004
trends/). Approximately 36% of the increase in atmospheric CO2 is a direct result of the combustion of petroleum for transportation purposes [2]. The potential environmental consequences [2] have led to an urgent need to develop renewable, carbon-neutral, fuels that can directly displace petroleum. Alga-based fuels potentially meet these criteria. However to date, their market penetration has been negligible. We review the potential for a specific algal taxon, diatoms, to become the biofuel of the future. Diatoms are major sources of fossil fuels Diatoms (see Glossary) are unicellular eukaryotic algae that entered the fossil record 150 million years ago [3]. They are secondary symbionts, distinguished from most other algal forms by possessing a siliceous shell, or frustule. They rose to ecological prominence 34 million years ago in the Oligocene, with the opening of the Drake Passage and subsequent global cooling [4]. Their ecological success introduced a major source of organic carbon for marine food webs, leading to the formation of massive fisheries – including whales [5]. A significant portion of diatom blooms sink along continental margins and shallow seas. Over geological time, a small fraction of this sinking flux was converted to petroleum. In the past 70 years, geochemists proved that algal lipids are the major feedstock for petroleum. The source of the algae can often be traced from analysis of lipids that act as biomarkers, and are stable over several million years in the petroleum reservoir [6]. The main biomarkers for
Glossary Aquatic Species Program (ASP): the first comprehensive project to estimate the potential of algae as biofuel feedstock, run by the US Department of Energy (DOE) from 1978 to 1996 with an overall investment of more than $25 million USD. Most studies focused on induction of lipid production in the tested strains under different environmental conditions. Biologically based renewable fuels (biofuels): alternative fuel sources based on converting living organisms to fuel within a short to intermediate time scale. Diatoms: unicellular organisms that constitute one of the major lineages of photosynthetic eukaryotes on earth. There are about 105 species of diatoms ranging in size between 4 and 200 mm. They have high productivity rate, outcompete other phytoplankters, have high environmental flexibility, and known to be highly resilient to many biotic and abiotic factors. They store energy in the form of triacylglycerols. Photosynthetic saturation point: the light intensity in which the rate of O2 evolution reaches a plateau. Beyond this point, any excessive photons will not be used for photochemistry. Transesterification: a chemical reaction between oils (TAG) and an alcohol (commonly methanol, ethanol, propanol, or butanol) to produce glycerol and alkyl esters of fatty acids, the latter are known as biodiesel. Triacylglycerol (TAG): made from three fatty acids and one glycerol. The main storage component in many algae, including diatoms. Trends in Biotechnology, March 2014, Vol. 32, No. 3
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(A)
C28 C30
C25
C29 (B)
R3
O
O
OH O
O
R2
HO –– CH3
+
O
R1
O
OH
OH
R1 OH
HO –– CH3
+
O R2
HO –– CH3
H
OH
OH
O R3
O
Triacylglycerol
Methanol (alcohol)
Glycerol
Methyl esters
1.5 × 10–06
TAG accumulaon (nmol TAG d–1)
(C)
Staonary phase
1.0 × 10–06
5.0 × 10–07 Exponenal phase
0 0.0
0.5
1.0
Growth rate (d–1) (D)
Exponenal phase
Staonary phase
10 µm
10 µm TRENDS in Biotechnology
Figure 1. Diatom lipid characteristics. (A) Chemical structure of diatoms biomarkers: C25 HBI, C30 HBI, and the ratio of the steranes C28/C29. (B) Chemical structure of TAG and its conversion to fatty acid methyl esters. (C) Accumulation of TAG versus the changes in growth rates during growth of Phaeodactylum tricornutum under nitrogen replete-starting conditions. (D) Fluorescence and light microscopy of lipid bodies in P. tricornutum at exponential and stationary phases – red fluorescence is chlorophyll autofluorescence and green fluorescence staining of natural lipid with BODIPY (493/503) dye. Abbreviations: BODIPY, boron-dipyrromethene; HBI, highly branched isoprenoids; TAG, triacylglycerol.
diatoms in petroleum are the ratio of steranes containing 28 and 29 carbon atoms [7,8], 24-norcholestanes [9,10], and highly branched isoprenoid (HBI) alkenes (Figure 1A) [11]. These biomarkers are found in many of the highest quality oil fields around the world. 118
Targeting diatom lipids for biofuel production To date, biofuels have been clustered into four generations of innovation. The first and second generations are based on higher plant oils, which can satisfy only a small fraction of the existing demand for transportation fuels, without
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Box 1. Are diatom lipids suitable for biodiesel production? Diatoms predominantly produce 13–21-carbon FAs, mainly composed of saturated and monounsaturated FAs (MUFAs) (Table I) that yield higher energy upon oxidation when compared to polyunsaturated FAs (PUFAs) with the same amount of carbons. The two predominant FAs are 16:1 and 16:0 in marine and freshwater diatoms; together they constitute up to 70% of the lipid profile of the cell. 14:0 FAs can account for up to 32% of the total lipids of the cell, depending on growth conditions [65] (Table I). The fourth most common FA is eicosapentaenoic acid (EPA, 20:5, v-3), which is widely known for its economic importance in the food and health industries. Biodiesel properties are directly influenced by the properties of the FAs from which they are made. The length and saturation level of the FAs have different effects on biodiesel parameters such as cetane
number, level of emissions, cold flow, oxidative stability, viscosity, and lubricity [66,67]. Biodiesel characteristics often have opposite requirements, for example, the presence of PUFAs improves the coldtemperature properties of biodiesel but reduces its oxidative stability while increasing its nitrogen oxide (NOx) emissions. It appears that no natural FA profile can suffice the production of an ideal biodiesel, yet the use of feedstock with high levels of MUFA, such as palmitoleate (16:1) or oleate (18:1), and low levels of saturated FA and PUFA, could produce biodiesel with close to optimal characteristics [66,68,69]. Optimizing the FA profile could increase their economic value. Implementation of metabolic engineering tools could allow this modification, as was done with soybeans with increased levels of oleic acid [70,71].
Table I. FAs in 17 different diatom species under various culture and environmental conditions Organism Phaeodactylum tricornutum Thallasiosira psuedonana Chaetoceros sp. Navicula inserta Navicula muralis Navicula pelliculosa Nitzschia closterium Nitzschia palea Nitzschia closterium Nitzschia longissima Nitzschia ovalis Nitzschia frustulum Cyclotella cryptica Amphora exigua Amphora sp. Biddulphia aurica Fragilaria sp.
Predominant fatty acids (>10%) 16:0, 16:1, 20:5 14:0, 16:12, 20:5 14:0, 16:1 16:0, 16:1, 20:5 16:0, 16:1 16:1, 20:5 16:0, 16:1, 20:5 16:0, 16:1, 20:5 16:1, 20:5 16:0, 16:1 16:0, 16:1 16:0, 16:1 16:0, 16:1, 16:3, 20:5 16:0, 16:1 16:0, 16:1, 20:5 14:0, 16:1, 20:5 14:0, 16:1, 20:5
competing with crops for food and other resources [12,13]. The third and fourth generation biofuels are based on algae, and have received a great deal of attention in the past 50 years. Diatoms are extremely successful in the contemporary oceans. They often outcompete other algae in mixed cultures [14,15] and are relatively resistant to pathogens [16,17]. Their major carbon storage product is lipids, especially triacylglycerides (TAGs) [18], and under normal growth conditions, between 15 and 25% of their biomass is composed of fatty acids (Box 1). In the early 1980s, the Aquatic Species Program, under the auspices of the US Department of Energy (ASP, http://www.nrel.gov/biomass/ pdfs/24190.pdf) screened 3000 algal strains for their potential to produce lipids. Of these, 50 strains were identified as worthy of consideration for commercial production; 60% of these were diatoms [19–21]. In fact, in a survey of 30 species of algae, it was found that diatoms could reach an average of 25% lipids/dry weight during exponential growth; 8% higher than green algae [22]. A comparison of the strengths and weaknesses of green algae and diatoms for biofuel production was reviewed by Hildebrand et al. [23]. Based on their lipid profile [20] and physiological characteristics, diatoms clearly are an underexploited, underappreciated biotechnological target for biofuel production (Box 1).
Other fatty acids (1–10%) 14:0, 16:2, 16:3, 18:1, 18:2 16:0, 16:2, 16:3, 18:1, 18:2 16:0, 18:1, 16:3, 20:4, 20:5, 14:0, 16:2, 16:3, 18:2 14:0, 16:2, 16:3, 18:2, 20:4, 14:0, 16:0, 16:2, 16:3, 18:1, 14:0, 16:3, 18:1, 18:2 14:0, 16:2, 16:3, 18:2, 20:4 14:0, 16:0, 16:2, 16:3, 18:1, 14:0, 16:3, 18:1, 18:2, 20:4, 14:0, 16:2, 16:3, 18:1, 18:2, 14:0, 16:2, 16:3, 18:1, 20:5 14:0, 22:6 14:0, 16:2, 16:3, 18:2, 18:1, 14:0, 16:2, 16:3, 18:1 16:0, 16:2, 16:3 16:0, 16:2, 16:3, 18:2
22:6 20:5 18:2
18:2 20:5 20:4, 20:5
20:5
Refs [65] [65] [72] [73] [73] [65] [74] [73] [65] [65] [65] [65] [44] [65] [65] [65] [65]
The scaling problem in displacing petroleum The physical and chemical properties of algal biodiesel are similar to petroleum-based diesel fuels (the latter being derived from the former), and thus require little or no modifications for use in conventional engines [24–26]. Yet, to be economically competitive with fossil petroleum, there are major hurdles to be overcome, starting with identifying the best strains through optimizing cultivation, harvesting, extracting, and refining. Engineering solutions to these problems are required to lower the production cost per barrel of algal biofuel from the estimated $300 or more to compete with the petroleum at $100 per barrel currently on the world market. In the USA alone, petroleum consumption is 20 million barrels of oil per day (http:// www.eia.gov/). Is it feasible to displace the demand with an alga-based fuel? From an economic perspective, the difference between extraction and refining petroleum and the production of biodiesel from algal mass cultures is in efficiency. At most, 0.1% of algal biomass enters the sediments of shallow seas and continental margins [27]. Of this, between 0.001 and 0.0001% becomes incorporated into a petroleum reservoir over geological time [28,29]. Although this deposition rate is extremely low, over geological time huge reservoirs of petroleum have formed. Each year, humans extract 1 million years worth of accumulated oil. To match that source 119
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Box 2. Biomass productivity of diatoms grown in outdoor systems Figure I summarizes experimentally measured and calculated productivity of marine unicellular algae including diatoms [14,21,58,75–79]. The data were derived from natural field conditions and outdoor systems. In Florida, outdoor ponds filled with wastewater–seawater mixtures yielded a productivity of 15–24 g/m2/d (corresponding to 1–1.7 g C/m/d) [14]. In those experiments, monocultures of marine diatoms (Phaeodactylum tricornutum, Amphiprora sp., amphora sp., and Nitzschia closterium) were formed by outcompeting the other algae present in the system at inoculation [14]. An ASP study revealed that, while grown in outdoor ponds, the productivity of Amphora, Chaetoceros muelleri, and Cyclotella cryptica was 26–39 g m2 d1 (Figure I) with a lipid content ranging between 24 and 40% of dry weight [21]. In addition, growing Cyclotella sp. in outdoor ponds in Fort Pierce, Florida yielded an average biomass of 20 g/m2/d [58] (Figure I). Thus,
given sufficient light, diatoms grown in ponds can easily reach a productivity of 17 g/m2/d or higher which is the calculated threshold required to achieve a positive EROI [62]. This places diatom productivity at upper range of that reported for outdoor open pond systems [62]. To compute the EROI we used the following equation: P energyOUTþ aj oj EROI ¼ energyINþP j 2 Jb I , where: energyIN is direct energy flows k2K k k
including electricity and fuel consumed during production; energyOUT is biofuel produced; K is non-energy inputs where k2K; J is nonenergy outputs, where j2J; Ik is quantity of the kth non-energy input; bk is per-unit energy equivalent value of the kth input; oj is quantity of the jth non-energy output; aj is the per-unit energy equivalent value for the jth output.
Benemann and Oswald, 1996
Biomass producvity from system design costs and producvies for outdoor pond
Weismann and Goebel 1987 Benemann et al., 1982 Huesemann et al., 2009
Measured biomass producvity of diatoms growing in outdoor ponds
Sheehan et al., 1998 Goldman and Ryther, 1975 Ryther, 1959 (high irradiance)
Calculated producvity of marine phytoplakton
Ryther, 1959 (mid irradiance) 0
10 20 30 Biomass producvity (g/m2 /d1)
40 TRENDS in Biotechnology
Figure I. Biomass productivity of oceanic algae grown in outdoor ponds – Data are taken from the different studies noted on the y axis and are presented as values of g/ m2/d. The gray dashed line at x = 17 represents the lowest value to achieve a EROI >1 for biofuel production [62].
requires a highly efficient production system; nevertheless, the task is far from impossible. For heuristic purposes we can make the calculations with diatoms as the feedstock. Gallagher [30] calculated that for algae to be economically viable, the system productivity should be in the range of 100 mt/ha/year. The published productivity data for diatoms ranges between 8 and 39 g/m2/day (Box 2), which corresponds to 29–142 mt/ha/year, or 0.6–2.7 g C/m2/day in diatoms. Based on these values, we calculate that diatom production in outdoor ponds can meet the defined baseline targets [30] (Box 2, Table 1). Moreover, taking into consideration the mid and high productivity values of diatoms (Table 2) and a lipid content of 35% by weight (see Table S1 in the supplementary material online) [30], we calculate that the annual yield of diatom-based biodiesel can approach 9000–15 000 gallons per hectare (Table 1). Will this be enough to supply the USA consumption demands? According to theoretical biodiesel yield calculations, it appears that 100% of the present demand for oil in the USA could be met using only 3% of USA land area. Is that physically possible? The limiting factor that physically limits the production of alga-based biofuels is light. Given an average of 31025 photosynthetically active quanta/ m2/day incident within 358 latitude on either side of the equator, and assuming a modest photosynthetic energy conversion efficiency of 3%, with 35% of the photosynthetic product directed toward lipids, the system would produce 120
1 g C/m2/day. Given that the daily rate of petroleum consumption in the USA is 241011 g C/day, the production of diatom lipids would require 5% of the land area of the USA. These calculations clearly demonstrate the potential efficiency of diatoms as a source of carbon-neutral transportation fuels. Boosting lipid production in diatoms using environmental manipulations The production of diatoms in photobioreactors, as well as indoor and outdoor ponds, has been demonstrated for >50 years [31–33], and large-scale culture of diatoms is used for feeding shrimp and mollusks in commercial aquaculture [34–36], but diatoms have been largly neglected as a biofuel feedstock. In the laboratory, many researchers have searched for the ‘sweet spot’ of controlling the ‘carbon decision tree’ for switching between biomass accumulation and lipid production in algae in general, and diatoms in particular (Box 3). We summarize the third and fourth generation attempts to increase lipid accumulation in a variety of marine and freshwater diatoms from 1965 to 2013 (Table S1). In general, when grown under different conditions, diatoms can accumulate between 25% and 45% lipids on a dry weight basis, which is remarkably high [20,37]. Nitrogen starvation leads to accumulation of total lipids and total fatty acids (TFAs) and increases the proportion of TAGs (Table S1) [38–41]. However, nitrogen starvation
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Table 1. Biodiesel yield calculation and land area required for growth based on published productivity values reported for diatoms growing in outdoor pondsa
Low diatom productivity (8 g/m2/yr) Medium diatom productivity (24 g/m2/yr) High diatom productivity (39 g/m2/yr)
Biomass productivity (mt/ha/yr)
Average lipid %
Biodiesel yield (gallons/ha/yr) 3022
Overall area in acres to reach 5 million barrels per day 46 239 606
Percentage of the USA land area needed to grow 5 million barrels per day 2.0
29
35
86
35
8962
15 592 425
0.7
142
35
14 798
9443 300
0.4
a
The biodiesel yield was calculated based on a conversion ratio obtained from Gallagher [30].
Table 2. Difference in dry weight and TAG yield for P. tricornutum during exponential and stationary growth phases
Biomass (dry weight) TAG
g/cell (•10S11)
g/L (•10S1)
exp 5.7 0.05
exp 0.57 0.005
stat 4.4 0.37
stat 4.4 0.37
inevitability leads to reduced growth rates and may not be the best third-generation approach to increase productivity of diatoms [23,37]. An alternative approach to enhancing lipid accumulation is silicon starvation. Diatoms accumulate TAG under silicon limitation without suffering from physiological damage observed under nitrogen starvation. A study on nine diatoms showed that silicon starvation increased their lipid content from 28 to 61%/dry weight, with an average of 4510% (Table S1). In fact, silicon starvation often leads to a higher accumulation of total lipids in diatoms than nitrogen starvation [18,21,23,41–44]. TAG accumulation in diatoms could also be enhanced when the cell cycle is arrested [45,46]. Lipids are primarily synthesized in the G1 phase of the cell division cycle. In Nitzschia palea, lipids accumulate in the presence of an autotoxin that blocks cell division [45]. A study done Box 3. Carbon decision tree The fate of photosynthetically fixed carbon is strongly influenced by environmental conditions. In diatoms, most photosynthetically fixed carbon flows through pyruvate, from which it is decarboxylated by the pyruvate dehydrogenase complex (PDC) to form acetyl-CoA (AcCoA). Under most growth conditions, most of the AcCoA will enter the tricarboxcylic acid (TCA) cycle to form intermediate metabolites for cellular anabolic pathways such as amino acid biosynthesis. However, when stressed, cells will divert their newly fixed carbon toward storage components, by irreversibly carboxylating the AcCoA to produce malonyl-CoA, which is the substrate for FA biosynthesis. In most diatoms, those storage components will be TAGs that accumulate in lipid bodies to serve as energy reservoirs. The commitment of AcCoA to either pathway is the heart of the carbon decision tree of the cell. Hence, it seems that there is an inevitable tradeoff between cell growth lipid content [79]. This tradeoff is one of the biggest hurdles in the industrial production of low-cost algal biodiesel [20]. TAG synthesis is favored when energy input exceeds the cellular capacity for utilizing the energy [46]. TAG biosynthesis is effectively an energy sink that serves as a photoprotective mechanism while simultaneously allowing the cell to store carbon. The production of TAGs is enhanced by a variety of stresses, including nutrient limitation, high light or high UV fluxes, high or low salt, and low pH, as well as cell cycle arrest.
Harvests year
per
g/l/year
Exp 120 120
stat 45 45
exp 6.84 0.06
mt/ha/year stat 19.80 1.65
exp 13.6 0.12
stat 39.6 3.3
in our group showed that arresting the cell cycle of Phaeodactylum tricornutum using a cyclin-dependent kinase 1 and 2 inhibitor increases the cellular TAG content by 14fold [47]. Thalassiosira pseudonana also exhibits rapid accumulation of lipids in the presence of a microtubulebased inhibitor [23]. Diatoms as fourth-generation biofuels Although third-generation strategies may prevail, genetic manipulation of cells is also possible. The aim of the socalled fourth-generation biofuel is to co-opt basic biochemical pathways by using molecular genetic tools to generate photoautotrophic algal strains with high lipid yield. To date, there is abundant literature on genetic manipulation of algae that focuses on green algae in general, and on Chlamydomonas reinhardtii in particular. However, it is unlikely that this freshwater green alga will be a commercial biofuel producer [48]. Although diatoms are diploid during normal growth, they do not have an easily controlled sexual recombination phase. Therefore, using breeding approaches and/or classical genetics to select for specific traits is not practical. However, genetic transformation of diatoms has been reported since the 1990s [19,49–52]. Annotated genomes of P. tricornutum and T. pseudonana are published [53,54], and raw sequence data from the genomes of Fragilariopsis cylindrus and Pseudo-nitzschia multiseries are available (http://genome.jgi.doe.gov/). Together with new information from flux balance and transcriptomic analyses, transformation techniques [50,55,56] can be used to increase diatom lipid production efficiency. Only a few studies describing genetic manipulation of diatoms to increase their biomass or lipid content have been published to date (Table S1). Insertion of acetyl-CoA carboxylase gene (acc1) to Cyclotella cryptica led to increased activity of the enzyme [19,21], and overexpression of two plant thioesterases (C14-TE and C12-TE) from Cinnamomum camphora increased the accumulation of short saturated chain length fatty acids (FAs) in P. tricornutum [57]. Yet, neither transformation increased the total cellular lipid 121
Opinion content nor yielded any significant increase in secretion of FA. Huesemann et al. [58] obtained a Cyclotella sp. strain with higher photosynthetic saturation point, however, no improvements in biomass productivities were observed. The engineering of P. tricornutum to uptake glucose by expressing the human glucose transporter glut1 led to a tenfold increase in cell density under heterotrophic growth conditions [59]. This may be promising from the standpoint of increasing biomass, however heterotrophic growth of algae for fuels defeats the purpose of obtaining a carbon-neutral fuel source. Recently, T. pseudonana with a multifunctional knockdown was lipase/phospholipase/acyltransferase shown to increase lipid yields without affecting growth [52]. Diatom-based fourth-generation biofuel is clearly in its infancy. Nonetheless, targeting nitrogen uptake, the glutamine synthetase/glutamine oxoglutarate aminotransferase pathway, and TAG biosynthesis appear to be promising directions for genetic manipulations [39,60,61]. We suggest that testing the alteration of those branch points while finding the best growth conditions for the obtained strains will bring us closer to mass production of biofuel. Finally, increased resistance to biotic stresses (competitors, parasites, and pathogens) by introduction of new genes (e.g., herbicides) could improve the sustainability of the system and further increase the Energy Return (On) Investment (EROI). Optimizing the efficiency of lipid production An objective metric of a production system is based on the concept of EROI, which is the ratio of the energy obtained to the amount of energy invested. Although there are large uncertainties at virtually all steps of the biofuel production process, an EROI higher than the break-even value of 1 requires an algal biomass productivity above 17 g/m/day, corresponding to 1.2 g C/m/day, in purely photosynthetic systems [62]. Energy costs could be influenced by managing nutrient supply, lipid extraction, and co-production of high-value compounds. To date, there are no EROI evaluations of any diatom feedstock, either in the laboratory or the field. Yet, manipulating the productivity of the production line will result in a higher EROI. In addition, the cost estimation of biofuel production depends on the number of harvests per year [63]. A comparison of exponential and stationary phase P. tricornutum cultures, in terms of their culture densities, dry weight, and lipid profile [38,39] (Figure 1), suggests that harvesting during stationary phase (on Day 8) versus exponential phase (Day 3) results in a threefold increase in biomass and a 38-fold increase in TAG (Table 2). Reducing the number of harvests can also increase the EROI by reducing the energy investment in two of the most expensive components of the production system: cultivation and harvesting. In addition to productivity per se, our analyses also show that the diversity of FA increased during stationary phase, changing from mostly 16:0 and 16:1 during exponential phase to include significant amounts of 14:0, 18:0, 18:2, and 20:5 during stationary phase. Concluding remarks and future perspectives A major portion of current high-quality petroleum is derived from fossilized diatoms. The rise of diatoms to 122
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ecological prominence over the past 34 million years is due to their high photosynthetic energy conversion efficiency and rapid uptake and assimilation of nutrients. It would be worthwhile to take advantage of the natural selection of these organisms that have evolved such high photosynthetic energy conversation with lipids as their primary storage product. Growing diatoms as feedstock for biofuel production could displace all petroleum consumption in the USA. However, it should be clear that the future of biofuels is based on a technological conundrum: the more one makes the biological product, the cheaper the fossil product will become. Consequently, the displacement of fossil fuels will require more than technological advances and investment in infrastructure; it will require strong market incentives, such as ‘wedge taxes’ that fix the costs of petroleum to match that of algal biofuels. Without policy intervention, the continued reliance on fossil fuels will further distort global climate systems, leading to a reduction in oceanic primary production [64] and in food supplies to higher trophic levels, including whales. Ironically, although the exploitation of petroleum may have saved whales 150 years ago, the increased use of oil as a fuel may ultimately lead to the demise of these and other animals in the coming century. Acknowledgments Our research is supported by the United States Department of Energy (DOE) Consortium of Algal Biofuels Commercialization (CAB-Comm) program, a gift from James G. Gibson to PGF, the Bennett L. Smith Endowment, and the Rutgers Energy Institute. We thank Benjamin Van Mooy for analyzing the TAG amounts and composition of our Phaeodactylum tricornutum cultures. We thank Robert Kopp for comments about life cycle assessments. Confocal microscopy to view lipid bodies was done at Rutgers University SEBS Core Facility and, funded by NIH1S10RR025424 to N. E. Tumer, with the assistance of Michael D. Pierce.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.tibtech.2014.01.004. References 1 Rodrigue, J-P. et al. (2013) Transportation and energy. In The Geography of Transport Systems. (3rd edn), Routledge 2 IPCC (2013) Climate Change 2013: The Physical Science Basis. In Working Group I Contribution to the IPCC 5th Assessment Report (AR5). United Nations 3 Schuhmann, H. et al. (2011) Perspectives on metabolic engineering for increased lipid contents in microalgae. Biofuels 3, 71–86 4 Khozin-Goldberg, I. and Cohen, Z. (2011) Unraveling algal lipid metabolism: recent advances in gene identification. Biochimie 93, 91–100 5 Berger, W.H. (2007) Cenozoic cooling, Antarctic nutrient pump, and the evolution of whales. Deep-Sea Res. PT II 54, 2399–2421 6 Brocks, J.J. and Summons, R.E. (2003) Sedimentary hydrocarbons, biomarkers for early life. Treatise Geochem. 8, 63–115 7 Grantham, P.J. and Wakefield, L.L. (1988) Variations in the sterane carbon number distributions of marine source rock derived crude oils through geological time. Org. Geochem. 12, 61–73 8 Katz, M.E. et al. (2004) Evolutionary trajectories and biogeochemical impacts of marine eukaryotic phytoplankton. Annu. Rev. Ecol. Evol. Syst. 35, 523–556 9 Holba, A.G. et al. (1998) 24-norcholestanes as age-sensitive molecular fossils. Geology 26, 783–786 10 Holba, A.G. et al. (1998) Application of 24-norcholestanes for constraining source age of petroleum. Org. Geochem. 29, 1269–1283
Opinion 11 Greenwell, H.C. et al. (2010) Placing microalgae on the biofuels priority list: a review of the technological challenges. J. R. Soc. Interface 7, 703–726 12 Chisti, Y. (2007) Biodiesel from microalgae. Biotechnol. Adv. 25, 294– 306 13 Chisti, Y. (2008) Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26, 126–131 14 Goldman, J.C. and Ryther, J.H. (1975) Mass production of marine algae in outdoor cultures. Nature 254, 594–595 15 Amano, Y. et al. (2012) Competition between the cyanobacterium Microcystis aeruginosa and the diatom Cyclotella sp. under nitrogenlimited condition caused by dilution in eutrophic lake. J. Appl. Phycol. 24, 965–971 16 Hamm, C. and Smetacek, V. (2007) Armor: why, when and how? In Evolution of Primary Producers in the Sea (Falkowski, P.G. and Knoll, A.H., eds), pp. 311–332, Academic Press 17 Tomaru, Y. et al. (2012) First evidence for the existence of pennate diatom viruses. ISME J. 6, 1445–1448 18 Taguchi, S. et al. (1987) Silicate deficiency and lipid-synthesis of marine diatoms. J. Phycol. 23, 260–267 19 Dunahay, T.G. et al. (1996) Manipulation of microalgal lipid production using genetic engineering. Appl. Biochem. Biotechnol. 57–58, 223–231 20 Hu, Q. et al. (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639 21 Sheehan, J. et al. (1998) A look back at the US Department of Energy’s Aquatic Species Program – biodiesel from algae, National Renewable Energy Laboratory 22 Horst, I. et al. (2012) Treatment of Phaeodactylum tricornutum cells with papain facilitates lipid extraction. J. Biotechnol. 162, 40–49 23 Hildebrand, M. et al. (2012) The place of diatoms in the biofuels industry. Biofuels 3, 221–240 24 Al-Zuhair, S. (2007) Production of biodiesel: possibilities and challenges. Biofuels Bioprod. Biofpr 1, 57–66 25 Liu, B. and Zhao, Z. (2007) Biodiesel production by direct methanolysis of oleaginous microbial biomass. J. Chem. Technol. Biotechnol. 82, 775– 780 26 Courchesne, N.M.D. et al. (2009) Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J. Biotechnol. 141, 31–41 27 Emerson, S. and Hedges, J.I. (1988) Processes controlling the organic carbon content of open ocean sediments. Paleoceanography 3, 621–634 28 Dukes, J.S. (2003) Burning buried sunshine: human consumption of ancient solar energy. Clim. Change 61, 31–44 29 Philp, R.P. (2005) Formation and geochemistry of oil and gas. In Sediments, Diagenesis, and Sedimentary Rocks (1st edn) (Mackenzie, F.T., ed.), pp. 223–256, Elsevier 30 Gallagher, B.J. (2011) The economics of producing biodiesel from algae. Renew. Energy 36, 158–162 31 Ansell, A.D. et al. (1963) Studies on the mass culture of Phaeodactylum. II. The growth of Phaeodactylum and other species in outdoor tanks. Limnol. Oceanogr. 8, 184–206 32 Laws, E.A. (1985) Shallow algal mass culture systems for the production of oils. In Solar Energy Research Institute, Hawaii University, (Honolulu) 33 Miron, A.S. et al. (2003) Shear stress tolerance and biochemical characterization of Phaeodactylum tricornutum in quasi steady-state continuous culture in outdoor photobioreactors. Biochem. Eng. J. 16, 287–297 34 Coutteau, P. (1996) Microalgae. In Manual on the Production and Use of Live Food for Aquaculture (Coutteau, P. and Sorgeloos, P., eds), pp. 7–48, Food and Agriculture Organization of the United Nations 35 Lopez-Elias, J.A. et al. (2005) Indoor and outdoor mass production of the diatom Chaetoceros muelleri in a Mexican commercial hatchery. Aquacult. Eng. 33, 181–191 36 Turpin, V. et al. (2001) Oyster greening by outdoor mass culture of the diatom Haslea ostrearia Simonsen in enriched seawater. Aquac. Res. 32, 801–809 37 Frada, M.J. et al. (2013) Quantum requirements for growth and fatty acid biosynthesis in the marine diatom Phaeodactylum tricornutum (Bacillariophyceae) in nitrogen replete and limited conditions. J. Phycol. 49, 381–388 38 Alonso, D.L. et al. (2000) Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum. Phytochemistry 54, 461–471
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39 Guerra, L.T. et al. (2013) Regulatory branch points affecting protein and lipid biosynthesis in the diatom Phaeodactylum tricornutum. J. Biomass Bioenergy http://dx.doi.org/10.1016/j.biombioe.2013.1010.1007 40 Jiang, Y.L. et al. (2012) Photosynthetic performance, lipid production and biomass composition in response to nitrogen limitation in marine microalgae. Plant Physiol. Biochem. 54, 70–77 41 Shifrin, N.S. and Chisholm, S.W. (1981) Phytoplankton lipids: interspecific differences and effects of nitrate, silicate and light-dark cycles. J. Phycol. 17, 374–384 42 Coombs, J. et al. (1967) Studies on the biochemistry and fine structure of silica shell formation in diatoms. Chemical composition of Navicula pelliculosa during silicon-starvation synchrony. Plant Physiol. 42, 1601–1606 43 Lynn, S.G. et al. (2000) Effect of nutrient availability on the biochemical and elemental stoichiometry in the freshwater diatom Stephanodiscus minutulus (Bacillariophyceae). J. Phycol. 36, 510–522 44 Roessler, P.G. (1988) Effects of silicon deficiency on lipid-composition and metabolism in the diatom Cyclotella cryptica. J. Phycol. 24, 394– 400 45 Collyer, M. and Fogg, G.E. (1955) Studies on fat accumulation by algae. J. Exp. Bot. 6, 256–275 46 Roessler, P.G. (1990) Environmental control of glycerolipid metabolism in microalgae: commercial implications and future-research directions. J. Phycol. 26, 393–399 47 Falkowski, G.P. and Kim, J. Compositions and methods for enhancing lipid production in microalgae via induction of cell, WO/2013/028952, Rutgers University 48 Rasala, B. et al. (2013) Genetic engineering to improve algal biofuels production. In Algae for Biofuels and Energy (Borowitzka, M.A. and Moheimani, N.R., eds), pp. 99–113, Netherlands, Springer 49 Apt, K.E. et al. (1996) Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol. Gen. Genet. 252, 572–579 50 Falciatore, A. et al. (1999) Transformation of nonselectable reporter genes in marine diatoms. Mar. Biotechnol. (N.Y.) 1, 239–251 51 Poulsen, N. and Kroger, N. (2005) A new molecular tool for transgenic diatoms – control of mRNA and protein biosynthesis by an inducible promoter-terminator cassette. FEBS J. 272, 3413–3423 52 Trentacoste, E.M. et al. (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proc. Natl. Acad. Sci. U.S.A. http://dx.doi.org/10.1073/pnas.1309299110 53 Allen, A.E. et al. (2011) Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Nature 473, 203–209 54 Armbrust, E.V. et al. (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306, 79–86 55 Poulsen, N. et al. (2006) Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). J. Phycol. 42, 1059– 1065 56 De Riso, V. et al. (2009) Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res. 37, e96 57 Radakovits, R. et al. (2011) Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum. Metab. Eng. 13, 89–95 58 Huesemann, M.H. et al. (2009) Biomass productivities in wild type and pigment mutant of Cyclotella sp. (diatom). Appl. Biochem. Biotechnol. 157, 507–526 59 Zaslavskaia, L.A. et al. (2001) Trophic obligate conversion of a photoautotrophic organism through metabolic engineering. Science 292, 2073–2075 60 Valenzuela, J. et al. (2012) Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum. Biotechnol. Biofuels 5, 40 http://dx.doi.org/10.1186/1754-6834-1185-1140 61 Yang, Z.K. et al. (2013) Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation. Biotechnol. Biofuels 6, 67 62 Sills, D.L. et al. (2013) Quantitative uncertainty analysis of life cycle assessment for algal biofuel production. Environ. Sci. Technol. 47, 687– 694 63 Sun, A. et al. (2011) Comparative cost analysis of algal oil production for biofuels. Energy 36, 5169–5179 64 Behrenfeld, M.J. et al. (2006) Climate-driven trends in contemporary ocean productivity. Nature 444, 752–755 65 Orcutt, D.M. and Patterson, G.W. (1975) Sterol, fatty acid and elemental composition of diatoms grown in chemically defined media. Comp. Biochem. Physiol. B 50, 579–583 123
Opinion 66 Knothe, G. (2005) Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process. Technol. 86, 1059– 1070 67 Durrett, T.P. et al. (2008) Plant triacylglycerols as feedstocks for the production of biofuels. Plant J. 54, 593–607 68 McCormick, R.L. et al. (2001) Impact of biodiesel source material and chemical structure on emissions of criteria pollutants from a heavyduty engine. Environ. Sci. Technol. 35, 1742–1747 69 Knothe, G. and Dunn, R.O. (2003) Dependence of oil stability index of fatty compounds on their structure and concentration and presence of metals. J. Am. Oil Chem. Soc. 80, 1021–1026 70 Tat, M.E. et al. (2007) Exhaust emissions from an engine fueled with biodiesel from high-oleic soybeans. J. Am. Oil Chem. Soc. 84, 865– 869 71 Buhr, T. et al. (2002) Ribozyme termination of RNA transcripts downregulate seed fatty acid genes in transgenic soybean. Plant J. 30, 155– 163 72 Renaud, S.M. et al. (2002) Effect of temperature on growth, chemical composition and fatty acid composition of tropical Australian microalgae grown in batch cultures. Aquaculture 211, 195–214
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73 Opute, F.I. (1974) Lipid and fatty-acid composition of diatoms. J. Exp. Bot. 25, 823–835 74 Orcutt, D.M. and Patterson, G.W. (1974) Effect of light intensity upon lipid composition of Nitzschia closterium (Cylindrotheca fusiformis). Lipids 9, 1000–1003 75 Ryther, J.H. (1959) Potential productivity of the sea. Science 130, 602– 608 76 Benemann, J.R. et al. (1982) Microalgae as a source of liquid fuels. In Proceedings of the June 1982 SERI Biomass Program Principal Investigators’ Review Meeting, Aquatic Species Program Reports 77 Benemann, J.R. and Oswald, W.J. (1996) Systems and economic analysis of microalgae ponds for conversion of CO2 to biomass. Final report. No. DOE/PC/93204–T5. California Univ., Berkeley, CA (United States). Dept. of Civil Engineering 78 Weissman, J.C. and Goebel R.P. (1987) Design and analysis of microalgal open pond systems for the purpose of producing fuels: a subcontract report. No. SERI/STR-231-2840. Golden, CO (USA), Solar Energy Research Inst. 79 Falkowski, P.G. and Raven, J.A. (2007) Aquatic Photosynthesis, Princeton University Press
Diatoms: a fossil fuel of the future Orly Levitan1, Jorge Dinamarca1, Gal Hochman2, and Paul G. Falkowski1,3 1
Environmental Biophysics and Molecular Ecology Program, Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ 08901, USA 2 Department of Agriculture, Food & Resource Economics, Rutgers University, New Brunswick, NJ 08901, USA 3 Department of Earth and Planetary Sciences, Rutgers University, Piscataway, NJ 0885, USA
Long-term global climate change, caused by burning petroleum and other fossil fuels, has motivated an urgent need to develop renewable, carbon-neutral, economically viable alternatives to displace petroleum using existing infrastructure. Algal feedstocks are promising candidate replacements as a ‘drop-in’ fuel. Here, we focus on a specific algal taxon, diatoms, to become the fossil fuel of the future. We summarize past attempts to obtain suitable diatom strains, propose future directions for their genetic manipulation, and offer biotechnological pathways to improve yield. We calculate that the yields obtained by using diatoms as a production platform are theoretically sufficient to satisfy the total oil consumption of the US, using between 3 and 5% of its land area. The need for carbon-neutral fuels The first major oil well, drilled in 1859 by Edwin Drake, supplied cheap fuel for kerosene lamps, and led to a dramatic reduction in the demand for whale blubber. Although the use of kerosene as a fuel for lighting can be claimed as saving whales from being hunted to extinction, there were other unintended consequences to follow. The subsequent invention of internal combustion engines provided a huge demand for gasoline, which previously had been a worthless byproduct of kerosene distillation. By the early decades of the 20th century, the oil industry had become the engine of economic growth in industrializing nations. A legacy of Drake’s oil well is that over 150 years later, 96% of all transportation processes in the world is still based on petroleum [1]. The proven global reserves are projected to be able to meet the predicted demand for several decades (http://energy.gov/fe/ services/petroleum-reserves). However, an unintended consequence of the rapid combustion of fossil fuels is the rise in greenhouse gas emissions. Since the beginning of the Industrial Revolution, >350 billion metric tons of carbon have been emitted into the atmosphere, with a commitment rise in atmospheric CO2 of 43% over the past 150 years (http:// petrolog.typepad.com/climate_change/2010/01/cumulativeemissions-of-co2.html, http://www.esrl.noaa.gov/gmd/ccgg/ Corresponding authors: Levitan, O. ([email protected]); Dinamarca, J. ([email protected]). Keywords: biofuel; diatoms; lipids; biomass; productivity; Energy Return (On) Investment. 0167-7799/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.01.004
trends/). Approximately 36% of the increase in atmospheric CO2 is a direct result of the combustion of petroleum for transportation purposes [2]. The potential environmental consequences [2] have led to an urgent need to develop renewable, carbon-neutral, fuels that can directly displace petroleum. Alga-based fuels potentially meet these criteria. However to date, their market penetration has been negligible. We review the potential for a specific algal taxon, diatoms, to become the biofuel of the future. Diatoms are major sources of fossil fuels Diatoms (see Glossary) are unicellular eukaryotic algae that entered the fossil record 150 million years ago [3]. They are secondary symbionts, distinguished from most other algal forms by possessing a siliceous shell, or frustule. They rose to ecological prominence 34 million years ago in the Oligocene, with the opening of the Drake Passage and subsequent global cooling [4]. Their ecological success introduced a major source of organic carbon for marine food webs, leading to the formation of massive fisheries – including whales [5]. A significant portion of diatom blooms sink along continental margins and shallow seas. Over geological time, a small fraction of this sinking flux was converted to petroleum. In the past 70 years, geochemists proved that algal lipids are the major feedstock for petroleum. The source of the algae can often be traced from analysis of lipids that act as biomarkers, and are stable over several million years in the petroleum reservoir [6]. The main biomarkers for
Glossary Aquatic Species Program (ASP): the first comprehensive project to estimate the potential of algae as biofuel feedstock, run by the US Department of Energy (DOE) from 1978 to 1996 with an overall investment of more than $25 million USD. Most studies focused on induction of lipid production in the tested strains under different environmental conditions. Biologically based renewable fuels (biofuels): alternative fuel sources based on converting living organisms to fuel within a short to intermediate time scale. Diatoms: unicellular organisms that constitute one of the major lineages of photosynthetic eukaryotes on earth. There are about 105 species of diatoms ranging in size between 4 and 200 mm. They have high productivity rate, outcompete other phytoplankters, have high environmental flexibility, and known to be highly resilient to many biotic and abiotic factors. They store energy in the form of triacylglycerols. Photosynthetic saturation point: the light intensity in which the rate of O2 evolution reaches a plateau. Beyond this point, any excessive photons will not be used for photochemistry. Transesterification: a chemical reaction between oils (TAG) and an alcohol (commonly methanol, ethanol, propanol, or butanol) to produce glycerol and alkyl esters of fatty acids, the latter are known as biodiesel. Triacylglycerol (TAG): made from three fatty acids and one glycerol. The main storage component in many algae, including diatoms. Trends in Biotechnology, March 2014, Vol. 32, No. 3
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(A)
C28 C30
C25
C29 (B)
R3
O
O
OH O
O
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+
O
R1
O
OH
OH
R1 OH
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+
O R2
HO –– CH3
H
OH
OH
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O
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Methanol (alcohol)
Glycerol
Methyl esters
1.5 × 10–06
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5.0 × 10–07 Exponenal phase
0 0.0
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Figure 1. Diatom lipid characteristics. (A) Chemical structure of diatoms biomarkers: C25 HBI, C30 HBI, and the ratio of the steranes C28/C29. (B) Chemical structure of TAG and its conversion to fatty acid methyl esters. (C) Accumulation of TAG versus the changes in growth rates during growth of Phaeodactylum tricornutum under nitrogen replete-starting conditions. (D) Fluorescence and light microscopy of lipid bodies in P. tricornutum at exponential and stationary phases – red fluorescence is chlorophyll autofluorescence and green fluorescence staining of natural lipid with BODIPY (493/503) dye. Abbreviations: BODIPY, boron-dipyrromethene; HBI, highly branched isoprenoids; TAG, triacylglycerol.
diatoms in petroleum are the ratio of steranes containing 28 and 29 carbon atoms [7,8], 24-norcholestanes [9,10], and highly branched isoprenoid (HBI) alkenes (Figure 1A) [11]. These biomarkers are found in many of the highest quality oil fields around the world. 118
Targeting diatom lipids for biofuel production To date, biofuels have been clustered into four generations of innovation. The first and second generations are based on higher plant oils, which can satisfy only a small fraction of the existing demand for transportation fuels, without
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Box 1. Are diatom lipids suitable for biodiesel production? Diatoms predominantly produce 13–21-carbon FAs, mainly composed of saturated and monounsaturated FAs (MUFAs) (Table I) that yield higher energy upon oxidation when compared to polyunsaturated FAs (PUFAs) with the same amount of carbons. The two predominant FAs are 16:1 and 16:0 in marine and freshwater diatoms; together they constitute up to 70% of the lipid profile of the cell. 14:0 FAs can account for up to 32% of the total lipids of the cell, depending on growth conditions [65] (Table I). The fourth most common FA is eicosapentaenoic acid (EPA, 20:5, v-3), which is widely known for its economic importance in the food and health industries. Biodiesel properties are directly influenced by the properties of the FAs from which they are made. The length and saturation level of the FAs have different effects on biodiesel parameters such as cetane
number, level of emissions, cold flow, oxidative stability, viscosity, and lubricity [66,67]. Biodiesel characteristics often have opposite requirements, for example, the presence of PUFAs improves the coldtemperature properties of biodiesel but reduces its oxidative stability while increasing its nitrogen oxide (NOx) emissions. It appears that no natural FA profile can suffice the production of an ideal biodiesel, yet the use of feedstock with high levels of MUFA, such as palmitoleate (16:1) or oleate (18:1), and low levels of saturated FA and PUFA, could produce biodiesel with close to optimal characteristics [66,68,69]. Optimizing the FA profile could increase their economic value. Implementation of metabolic engineering tools could allow this modification, as was done with soybeans with increased levels of oleic acid [70,71].
Table I. FAs in 17 different diatom species under various culture and environmental conditions Organism Phaeodactylum tricornutum Thallasiosira psuedonana Chaetoceros sp. Navicula inserta Navicula muralis Navicula pelliculosa Nitzschia closterium Nitzschia palea Nitzschia closterium Nitzschia longissima Nitzschia ovalis Nitzschia frustulum Cyclotella cryptica Amphora exigua Amphora sp. Biddulphia aurica Fragilaria sp.
Predominant fatty acids (>10%) 16:0, 16:1, 20:5 14:0, 16:12, 20:5 14:0, 16:1 16:0, 16:1, 20:5 16:0, 16:1 16:1, 20:5 16:0, 16:1, 20:5 16:0, 16:1, 20:5 16:1, 20:5 16:0, 16:1 16:0, 16:1 16:0, 16:1 16:0, 16:1, 16:3, 20:5 16:0, 16:1 16:0, 16:1, 20:5 14:0, 16:1, 20:5 14:0, 16:1, 20:5
competing with crops for food and other resources [12,13]. The third and fourth generation biofuels are based on algae, and have received a great deal of attention in the past 50 years. Diatoms are extremely successful in the contemporary oceans. They often outcompete other algae in mixed cultures [14,15] and are relatively resistant to pathogens [16,17]. Their major carbon storage product is lipids, especially triacylglycerides (TAGs) [18], and under normal growth conditions, between 15 and 25% of their biomass is composed of fatty acids (Box 1). In the early 1980s, the Aquatic Species Program, under the auspices of the US Department of Energy (ASP, http://www.nrel.gov/biomass/ pdfs/24190.pdf) screened 3000 algal strains for their potential to produce lipids. Of these, 50 strains were identified as worthy of consideration for commercial production; 60% of these were diatoms [19–21]. In fact, in a survey of 30 species of algae, it was found that diatoms could reach an average of 25% lipids/dry weight during exponential growth; 8% higher than green algae [22]. A comparison of the strengths and weaknesses of green algae and diatoms for biofuel production was reviewed by Hildebrand et al. [23]. Based on their lipid profile [20] and physiological characteristics, diatoms clearly are an underexploited, underappreciated biotechnological target for biofuel production (Box 1).
Other fatty acids (1–10%) 14:0, 16:2, 16:3, 18:1, 18:2 16:0, 16:2, 16:3, 18:1, 18:2 16:0, 18:1, 16:3, 20:4, 20:5, 14:0, 16:2, 16:3, 18:2 14:0, 16:2, 16:3, 18:2, 20:4, 14:0, 16:0, 16:2, 16:3, 18:1, 14:0, 16:3, 18:1, 18:2 14:0, 16:2, 16:3, 18:2, 20:4 14:0, 16:0, 16:2, 16:3, 18:1, 14:0, 16:3, 18:1, 18:2, 20:4, 14:0, 16:2, 16:3, 18:1, 18:2, 14:0, 16:2, 16:3, 18:1, 20:5 14:0, 22:6 14:0, 16:2, 16:3, 18:2, 18:1, 14:0, 16:2, 16:3, 18:1 16:0, 16:2, 16:3 16:0, 16:2, 16:3, 18:2
22:6 20:5 18:2
18:2 20:5 20:4, 20:5
20:5
Refs [65] [65] [72] [73] [73] [65] [74] [73] [65] [65] [65] [65] [44] [65] [65] [65] [65]
The scaling problem in displacing petroleum The physical and chemical properties of algal biodiesel are similar to petroleum-based diesel fuels (the latter being derived from the former), and thus require little or no modifications for use in conventional engines [24–26]. Yet, to be economically competitive with fossil petroleum, there are major hurdles to be overcome, starting with identifying the best strains through optimizing cultivation, harvesting, extracting, and refining. Engineering solutions to these problems are required to lower the production cost per barrel of algal biofuel from the estimated $300 or more to compete with the petroleum at $100 per barrel currently on the world market. In the USA alone, petroleum consumption is 20 million barrels of oil per day (http:// www.eia.gov/). Is it feasible to displace the demand with an alga-based fuel? From an economic perspective, the difference between extraction and refining petroleum and the production of biodiesel from algal mass cultures is in efficiency. At most, 0.1% of algal biomass enters the sediments of shallow seas and continental margins [27]. Of this, between 0.001 and 0.0001% becomes incorporated into a petroleum reservoir over geological time [28,29]. Although this deposition rate is extremely low, over geological time huge reservoirs of petroleum have formed. Each year, humans extract 1 million years worth of accumulated oil. To match that source 119
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Box 2. Biomass productivity of diatoms grown in outdoor systems Figure I summarizes experimentally measured and calculated productivity of marine unicellular algae including diatoms [14,21,58,75–79]. The data were derived from natural field conditions and outdoor systems. In Florida, outdoor ponds filled with wastewater–seawater mixtures yielded a productivity of 15–24 g/m2/d (corresponding to 1–1.7 g C/m/d) [14]. In those experiments, monocultures of marine diatoms (Phaeodactylum tricornutum, Amphiprora sp., amphora sp., and Nitzschia closterium) were formed by outcompeting the other algae present in the system at inoculation [14]. An ASP study revealed that, while grown in outdoor ponds, the productivity of Amphora, Chaetoceros muelleri, and Cyclotella cryptica was 26–39 g m2 d1 (Figure I) with a lipid content ranging between 24 and 40% of dry weight [21]. In addition, growing Cyclotella sp. in outdoor ponds in Fort Pierce, Florida yielded an average biomass of 20 g/m2/d [58] (Figure I). Thus,
given sufficient light, diatoms grown in ponds can easily reach a productivity of 17 g/m2/d or higher which is the calculated threshold required to achieve a positive EROI [62]. This places diatom productivity at upper range of that reported for outdoor open pond systems [62]. To compute the EROI we used the following equation: P energyOUTþ aj oj EROI ¼ energyINþP j 2 Jb I , where: energyIN is direct energy flows k2K k k
including electricity and fuel consumed during production; energyOUT is biofuel produced; K is non-energy inputs where k2K; J is nonenergy outputs, where j2J; Ik is quantity of the kth non-energy input; bk is per-unit energy equivalent value of the kth input; oj is quantity of the jth non-energy output; aj is the per-unit energy equivalent value for the jth output.
Benemann and Oswald, 1996
Biomass producvity from system design costs and producvies for outdoor pond
Weismann and Goebel 1987 Benemann et al., 1982 Huesemann et al., 2009
Measured biomass producvity of diatoms growing in outdoor ponds
Sheehan et al., 1998 Goldman and Ryther, 1975 Ryther, 1959 (high irradiance)
Calculated producvity of marine phytoplakton
Ryther, 1959 (mid irradiance) 0
10 20 30 Biomass producvity (g/m2 /d1)
40 TRENDS in Biotechnology
Figure I. Biomass productivity of oceanic algae grown in outdoor ponds – Data are taken from the different studies noted on the y axis and are presented as values of g/ m2/d. The gray dashed line at x = 17 represents the lowest value to achieve a EROI >1 for biofuel production [62].
requires a highly efficient production system; nevertheless, the task is far from impossible. For heuristic purposes we can make the calculations with diatoms as the feedstock. Gallagher [30] calculated that for algae to be economically viable, the system productivity should be in the range of 100 mt/ha/year. The published productivity data for diatoms ranges between 8 and 39 g/m2/day (Box 2), which corresponds to 29–142 mt/ha/year, or 0.6–2.7 g C/m2/day in diatoms. Based on these values, we calculate that diatom production in outdoor ponds can meet the defined baseline targets [30] (Box 2, Table 1). Moreover, taking into consideration the mid and high productivity values of diatoms (Table 2) and a lipid content of 35% by weight (see Table S1 in the supplementary material online) [30], we calculate that the annual yield of diatom-based biodiesel can approach 9000–15 000 gallons per hectare (Table 1). Will this be enough to supply the USA consumption demands? According to theoretical biodiesel yield calculations, it appears that 100% of the present demand for oil in the USA could be met using only 3% of USA land area. Is that physically possible? The limiting factor that physically limits the production of alga-based biofuels is light. Given an average of 31025 photosynthetically active quanta/ m2/day incident within 358 latitude on either side of the equator, and assuming a modest photosynthetic energy conversion efficiency of 3%, with 35% of the photosynthetic product directed toward lipids, the system would produce 120
1 g C/m2/day. Given that the daily rate of petroleum consumption in the USA is 241011 g C/day, the production of diatom lipids would require 5% of the land area of the USA. These calculations clearly demonstrate the potential efficiency of diatoms as a source of carbon-neutral transportation fuels. Boosting lipid production in diatoms using environmental manipulations The production of diatoms in photobioreactors, as well as indoor and outdoor ponds, has been demonstrated for >50 years [31–33], and large-scale culture of diatoms is used for feeding shrimp and mollusks in commercial aquaculture [34–36], but diatoms have been largly neglected as a biofuel feedstock. In the laboratory, many researchers have searched for the ‘sweet spot’ of controlling the ‘carbon decision tree’ for switching between biomass accumulation and lipid production in algae in general, and diatoms in particular (Box 3). We summarize the third and fourth generation attempts to increase lipid accumulation in a variety of marine and freshwater diatoms from 1965 to 2013 (Table S1). In general, when grown under different conditions, diatoms can accumulate between 25% and 45% lipids on a dry weight basis, which is remarkably high [20,37]. Nitrogen starvation leads to accumulation of total lipids and total fatty acids (TFAs) and increases the proportion of TAGs (Table S1) [38–41]. However, nitrogen starvation
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Table 1. Biodiesel yield calculation and land area required for growth based on published productivity values reported for diatoms growing in outdoor pondsa
Low diatom productivity (8 g/m2/yr) Medium diatom productivity (24 g/m2/yr) High diatom productivity (39 g/m2/yr)
Biomass productivity (mt/ha/yr)
Average lipid %
Biodiesel yield (gallons/ha/yr) 3022
Overall area in acres to reach 5 million barrels per day 46 239 606
Percentage of the USA land area needed to grow 5 million barrels per day 2.0
29
35
86
35
8962
15 592 425
0.7
142
35
14 798
9443 300
0.4
a
The biodiesel yield was calculated based on a conversion ratio obtained from Gallagher [30].
Table 2. Difference in dry weight and TAG yield for P. tricornutum during exponential and stationary growth phases
Biomass (dry weight) TAG
g/cell (•10S11)
g/L (•10S1)
exp 5.7 0.05
exp 0.57 0.005
stat 4.4 0.37
stat 4.4 0.37
inevitability leads to reduced growth rates and may not be the best third-generation approach to increase productivity of diatoms [23,37]. An alternative approach to enhancing lipid accumulation is silicon starvation. Diatoms accumulate TAG under silicon limitation without suffering from physiological damage observed under nitrogen starvation. A study on nine diatoms showed that silicon starvation increased their lipid content from 28 to 61%/dry weight, with an average of 4510% (Table S1). In fact, silicon starvation often leads to a higher accumulation of total lipids in diatoms than nitrogen starvation [18,21,23,41–44]. TAG accumulation in diatoms could also be enhanced when the cell cycle is arrested [45,46]. Lipids are primarily synthesized in the G1 phase of the cell division cycle. In Nitzschia palea, lipids accumulate in the presence of an autotoxin that blocks cell division [45]. A study done Box 3. Carbon decision tree The fate of photosynthetically fixed carbon is strongly influenced by environmental conditions. In diatoms, most photosynthetically fixed carbon flows through pyruvate, from which it is decarboxylated by the pyruvate dehydrogenase complex (PDC) to form acetyl-CoA (AcCoA). Under most growth conditions, most of the AcCoA will enter the tricarboxcylic acid (TCA) cycle to form intermediate metabolites for cellular anabolic pathways such as amino acid biosynthesis. However, when stressed, cells will divert their newly fixed carbon toward storage components, by irreversibly carboxylating the AcCoA to produce malonyl-CoA, which is the substrate for FA biosynthesis. In most diatoms, those storage components will be TAGs that accumulate in lipid bodies to serve as energy reservoirs. The commitment of AcCoA to either pathway is the heart of the carbon decision tree of the cell. Hence, it seems that there is an inevitable tradeoff between cell growth lipid content [79]. This tradeoff is one of the biggest hurdles in the industrial production of low-cost algal biodiesel [20]. TAG synthesis is favored when energy input exceeds the cellular capacity for utilizing the energy [46]. TAG biosynthesis is effectively an energy sink that serves as a photoprotective mechanism while simultaneously allowing the cell to store carbon. The production of TAGs is enhanced by a variety of stresses, including nutrient limitation, high light or high UV fluxes, high or low salt, and low pH, as well as cell cycle arrest.
Harvests year
per
g/l/year
Exp 120 120
stat 45 45
exp 6.84 0.06
mt/ha/year stat 19.80 1.65
exp 13.6 0.12
stat 39.6 3.3
in our group showed that arresting the cell cycle of Phaeodactylum tricornutum using a cyclin-dependent kinase 1 and 2 inhibitor increases the cellular TAG content by 14fold [47]. Thalassiosira pseudonana also exhibits rapid accumulation of lipids in the presence of a microtubulebased inhibitor [23]. Diatoms as fourth-generation biofuels Although third-generation strategies may prevail, genetic manipulation of cells is also possible. The aim of the socalled fourth-generation biofuel is to co-opt basic biochemical pathways by using molecular genetic tools to generate photoautotrophic algal strains with high lipid yield. To date, there is abundant literature on genetic manipulation of algae that focuses on green algae in general, and on Chlamydomonas reinhardtii in particular. However, it is unlikely that this freshwater green alga will be a commercial biofuel producer [48]. Although diatoms are diploid during normal growth, they do not have an easily controlled sexual recombination phase. Therefore, using breeding approaches and/or classical genetics to select for specific traits is not practical. However, genetic transformation of diatoms has been reported since the 1990s [19,49–52]. Annotated genomes of P. tricornutum and T. pseudonana are published [53,54], and raw sequence data from the genomes of Fragilariopsis cylindrus and Pseudo-nitzschia multiseries are available (http://genome.jgi.doe.gov/). Together with new information from flux balance and transcriptomic analyses, transformation techniques [50,55,56] can be used to increase diatom lipid production efficiency. Only a few studies describing genetic manipulation of diatoms to increase their biomass or lipid content have been published to date (Table S1). Insertion of acetyl-CoA carboxylase gene (acc1) to Cyclotella cryptica led to increased activity of the enzyme [19,21], and overexpression of two plant thioesterases (C14-TE and C12-TE) from Cinnamomum camphora increased the accumulation of short saturated chain length fatty acids (FAs) in P. tricornutum [57]. Yet, neither transformation increased the total cellular lipid 121
Opinion content nor yielded any significant increase in secretion of FA. Huesemann et al. [58] obtained a Cyclotella sp. strain with higher photosynthetic saturation point, however, no improvements in biomass productivities were observed. The engineering of P. tricornutum to uptake glucose by expressing the human glucose transporter glut1 led to a tenfold increase in cell density under heterotrophic growth conditions [59]. This may be promising from the standpoint of increasing biomass, however heterotrophic growth of algae for fuels defeats the purpose of obtaining a carbon-neutral fuel source. Recently, T. pseudonana with a multifunctional knockdown was lipase/phospholipase/acyltransferase shown to increase lipid yields without affecting growth [52]. Diatom-based fourth-generation biofuel is clearly in its infancy. Nonetheless, targeting nitrogen uptake, the glutamine synthetase/glutamine oxoglutarate aminotransferase pathway, and TAG biosynthesis appear to be promising directions for genetic manipulations [39,60,61]. We suggest that testing the alteration of those branch points while finding the best growth conditions for the obtained strains will bring us closer to mass production of biofuel. Finally, increased resistance to biotic stresses (competitors, parasites, and pathogens) by introduction of new genes (e.g., herbicides) could improve the sustainability of the system and further increase the Energy Return (On) Investment (EROI). Optimizing the efficiency of lipid production An objective metric of a production system is based on the concept of EROI, which is the ratio of the energy obtained to the amount of energy invested. Although there are large uncertainties at virtually all steps of the biofuel production process, an EROI higher than the break-even value of 1 requires an algal biomass productivity above 17 g/m/day, corresponding to 1.2 g C/m/day, in purely photosynthetic systems [62]. Energy costs could be influenced by managing nutrient supply, lipid extraction, and co-production of high-value compounds. To date, there are no EROI evaluations of any diatom feedstock, either in the laboratory or the field. Yet, manipulating the productivity of the production line will result in a higher EROI. In addition, the cost estimation of biofuel production depends on the number of harvests per year [63]. A comparison of exponential and stationary phase P. tricornutum cultures, in terms of their culture densities, dry weight, and lipid profile [38,39] (Figure 1), suggests that harvesting during stationary phase (on Day 8) versus exponential phase (Day 3) results in a threefold increase in biomass and a 38-fold increase in TAG (Table 2). Reducing the number of harvests can also increase the EROI by reducing the energy investment in two of the most expensive components of the production system: cultivation and harvesting. In addition to productivity per se, our analyses also show that the diversity of FA increased during stationary phase, changing from mostly 16:0 and 16:1 during exponential phase to include significant amounts of 14:0, 18:0, 18:2, and 20:5 during stationary phase. Concluding remarks and future perspectives A major portion of current high-quality petroleum is derived from fossilized diatoms. The rise of diatoms to 122
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ecological prominence over the past 34 million years is due to their high photosynthetic energy conversion efficiency and rapid uptake and assimilation of nutrients. It would be worthwhile to take advantage of the natural selection of these organisms that have evolved such high photosynthetic energy conversation with lipids as their primary storage product. Growing diatoms as feedstock for biofuel production could displace all petroleum consumption in the USA. However, it should be clear that the future of biofuels is based on a technological conundrum: the more one makes the biological product, the cheaper the fossil product will become. Consequently, the displacement of fossil fuels will require more than technological advances and investment in infrastructure; it will require strong market incentives, such as ‘wedge taxes’ that fix the costs of petroleum to match that of algal biofuels. Without policy intervention, the continued reliance on fossil fuels will further distort global climate systems, leading to a reduction in oceanic primary production [64] and in food supplies to higher trophic levels, including whales. Ironically, although the exploitation of petroleum may have saved whales 150 years ago, the increased use of oil as a fuel may ultimately lead to the demise of these and other animals in the coming century. Acknowledgments Our research is supported by the United States Department of Energy (DOE) Consortium of Algal Biofuels Commercialization (CAB-Comm) program, a gift from James G. Gibson to PGF, the Bennett L. Smith Endowment, and the Rutgers Energy Institute. We thank Benjamin Van Mooy for analyzing the TAG amounts and composition of our Phaeodactylum tricornutum cultures. We thank Robert Kopp for comments about life cycle assessments. Confocal microscopy to view lipid bodies was done at Rutgers University SEBS Core Facility and, funded by NIH1S10RR025424 to N. E. Tumer, with the assistance of Michael D. Pierce.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.tibtech.2014.01.004. References 1 Rodrigue, J-P. et al. (2013) Transportation and energy. In The Geography of Transport Systems. (3rd edn), Routledge 2 IPCC (2013) Climate Change 2013: The Physical Science Basis. In Working Group I Contribution to the IPCC 5th Assessment Report (AR5). United Nations 3 Schuhmann, H. et al. (2011) Perspectives on metabolic engineering for increased lipid contents in microalgae. Biofuels 3, 71–86 4 Khozin-Goldberg, I. and Cohen, Z. (2011) Unraveling algal lipid metabolism: recent advances in gene identification. Biochimie 93, 91–100 5 Berger, W.H. (2007) Cenozoic cooling, Antarctic nutrient pump, and the evolution of whales. Deep-Sea Res. PT II 54, 2399–2421 6 Brocks, J.J. and Summons, R.E. (2003) Sedimentary hydrocarbons, biomarkers for early life. Treatise Geochem. 8, 63–115 7 Grantham, P.J. and Wakefield, L.L. (1988) Variations in the sterane carbon number distributions of marine source rock derived crude oils through geological time. Org. Geochem. 12, 61–73 8 Katz, M.E. et al. (2004) Evolutionary trajectories and biogeochemical impacts of marine eukaryotic phytoplankton. Annu. Rev. Ecol. Evol. Syst. 35, 523–556 9 Holba, A.G. et al. (1998) 24-norcholestanes as age-sensitive molecular fossils. Geology 26, 783–786 10 Holba, A.G. et al. (1998) Application of 24-norcholestanes for constraining source age of petroleum. Org. Geochem. 29, 1269–1283
Opinion 11 Greenwell, H.C. et al. (2010) Placing microalgae on the biofuels priority list: a review of the technological challenges. J. R. Soc. Interface 7, 703–726 12 Chisti, Y. (2007) Biodiesel from microalgae. Biotechnol. Adv. 25, 294– 306 13 Chisti, Y. (2008) Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26, 126–131 14 Goldman, J.C. and Ryther, J.H. (1975) Mass production of marine algae in outdoor cultures. Nature 254, 594–595 15 Amano, Y. et al. (2012) Competition between the cyanobacterium Microcystis aeruginosa and the diatom Cyclotella sp. under nitrogenlimited condition caused by dilution in eutrophic lake. J. Appl. Phycol. 24, 965–971 16 Hamm, C. and Smetacek, V. (2007) Armor: why, when and how? In Evolution of Primary Producers in the Sea (Falkowski, P.G. and Knoll, A.H., eds), pp. 311–332, Academic Press 17 Tomaru, Y. et al. (2012) First evidence for the existence of pennate diatom viruses. ISME J. 6, 1445–1448 18 Taguchi, S. et al. (1987) Silicate deficiency and lipid-synthesis of marine diatoms. J. Phycol. 23, 260–267 19 Dunahay, T.G. et al. (1996) Manipulation of microalgal lipid production using genetic engineering. Appl. Biochem. Biotechnol. 57–58, 223–231 20 Hu, Q. et al. (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639 21 Sheehan, J. et al. (1998) A look back at the US Department of Energy’s Aquatic Species Program – biodiesel from algae, National Renewable Energy Laboratory 22 Horst, I. et al. (2012) Treatment of Phaeodactylum tricornutum cells with papain facilitates lipid extraction. J. Biotechnol. 162, 40–49 23 Hildebrand, M. et al. (2012) The place of diatoms in the biofuels industry. Biofuels 3, 221–240 24 Al-Zuhair, S. (2007) Production of biodiesel: possibilities and challenges. Biofuels Bioprod. Biofpr 1, 57–66 25 Liu, B. and Zhao, Z. (2007) Biodiesel production by direct methanolysis of oleaginous microbial biomass. J. Chem. Technol. Biotechnol. 82, 775– 780 26 Courchesne, N.M.D. et al. (2009) Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J. Biotechnol. 141, 31–41 27 Emerson, S. and Hedges, J.I. (1988) Processes controlling the organic carbon content of open ocean sediments. Paleoceanography 3, 621–634 28 Dukes, J.S. (2003) Burning buried sunshine: human consumption of ancient solar energy. Clim. Change 61, 31–44 29 Philp, R.P. (2005) Formation and geochemistry of oil and gas. In Sediments, Diagenesis, and Sedimentary Rocks (1st edn) (Mackenzie, F.T., ed.), pp. 223–256, Elsevier 30 Gallagher, B.J. (2011) The economics of producing biodiesel from algae. Renew. Energy 36, 158–162 31 Ansell, A.D. et al. (1963) Studies on the mass culture of Phaeodactylum. II. The growth of Phaeodactylum and other species in outdoor tanks. Limnol. Oceanogr. 8, 184–206 32 Laws, E.A. (1985) Shallow algal mass culture systems for the production of oils. In Solar Energy Research Institute, Hawaii University, (Honolulu) 33 Miron, A.S. et al. (2003) Shear stress tolerance and biochemical characterization of Phaeodactylum tricornutum in quasi steady-state continuous culture in outdoor photobioreactors. Biochem. Eng. J. 16, 287–297 34 Coutteau, P. (1996) Microalgae. In Manual on the Production and Use of Live Food for Aquaculture (Coutteau, P. and Sorgeloos, P., eds), pp. 7–48, Food and Agriculture Organization of the United Nations 35 Lopez-Elias, J.A. et al. (2005) Indoor and outdoor mass production of the diatom Chaetoceros muelleri in a Mexican commercial hatchery. Aquacult. Eng. 33, 181–191 36 Turpin, V. et al. (2001) Oyster greening by outdoor mass culture of the diatom Haslea ostrearia Simonsen in enriched seawater. Aquac. Res. 32, 801–809 37 Frada, M.J. et al. (2013) Quantum requirements for growth and fatty acid biosynthesis in the marine diatom Phaeodactylum tricornutum (Bacillariophyceae) in nitrogen replete and limited conditions. J. Phycol. 49, 381–388 38 Alonso, D.L. et al. (2000) Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum. Phytochemistry 54, 461–471
Trends in Biotechnology March 2014, Vol. 32, No. 3
39 Guerra, L.T. et al. (2013) Regulatory branch points affecting protein and lipid biosynthesis in the diatom Phaeodactylum tricornutum. J. Biomass Bioenergy http://dx.doi.org/10.1016/j.biombioe.2013.1010.1007 40 Jiang, Y.L. et al. (2012) Photosynthetic performance, lipid production and biomass composition in response to nitrogen limitation in marine microalgae. Plant Physiol. Biochem. 54, 70–77 41 Shifrin, N.S. and Chisholm, S.W. (1981) Phytoplankton lipids: interspecific differences and effects of nitrate, silicate and light-dark cycles. J. Phycol. 17, 374–384 42 Coombs, J. et al. (1967) Studies on the biochemistry and fine structure of silica shell formation in diatoms. Chemical composition of Navicula pelliculosa during silicon-starvation synchrony. Plant Physiol. 42, 1601–1606 43 Lynn, S.G. et al. (2000) Effect of nutrient availability on the biochemical and elemental stoichiometry in the freshwater diatom Stephanodiscus minutulus (Bacillariophyceae). J. Phycol. 36, 510–522 44 Roessler, P.G. (1988) Effects of silicon deficiency on lipid-composition and metabolism in the diatom Cyclotella cryptica. J. Phycol. 24, 394– 400 45 Collyer, M. and Fogg, G.E. (1955) Studies on fat accumulation by algae. J. Exp. Bot. 6, 256–275 46 Roessler, P.G. (1990) Environmental control of glycerolipid metabolism in microalgae: commercial implications and future-research directions. J. Phycol. 26, 393–399 47 Falkowski, G.P. and Kim, J. Compositions and methods for enhancing lipid production in microalgae via induction of cell, WO/2013/028952, Rutgers University 48 Rasala, B. et al. (2013) Genetic engineering to improve algal biofuels production. In Algae for Biofuels and Energy (Borowitzka, M.A. and Moheimani, N.R., eds), pp. 99–113, Netherlands, Springer 49 Apt, K.E. et al. (1996) Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol. Gen. Genet. 252, 572–579 50 Falciatore, A. et al. (1999) Transformation of nonselectable reporter genes in marine diatoms. Mar. Biotechnol. (N.Y.) 1, 239–251 51 Poulsen, N. and Kroger, N. (2005) A new molecular tool for transgenic diatoms – control of mRNA and protein biosynthesis by an inducible promoter-terminator cassette. FEBS J. 272, 3413–3423 52 Trentacoste, E.M. et al. (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proc. Natl. Acad. Sci. U.S.A. http://dx.doi.org/10.1073/pnas.1309299110 53 Allen, A.E. et al. (2011) Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Nature 473, 203–209 54 Armbrust, E.V. et al. (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306, 79–86 55 Poulsen, N. et al. (2006) Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). J. Phycol. 42, 1059– 1065 56 De Riso, V. et al. (2009) Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res. 37, e96 57 Radakovits, R. et al. (2011) Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum. Metab. Eng. 13, 89–95 58 Huesemann, M.H. et al. (2009) Biomass productivities in wild type and pigment mutant of Cyclotella sp. (diatom). Appl. Biochem. Biotechnol. 157, 507–526 59 Zaslavskaia, L.A. et al. (2001) Trophic obligate conversion of a photoautotrophic organism through metabolic engineering. Science 292, 2073–2075 60 Valenzuela, J. et al. (2012) Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum. Biotechnol. Biofuels 5, 40 http://dx.doi.org/10.1186/1754-6834-1185-1140 61 Yang, Z.K. et al. (2013) Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation. Biotechnol. Biofuels 6, 67 62 Sills, D.L. et al. (2013) Quantitative uncertainty analysis of life cycle assessment for algal biofuel production. Environ. Sci. Technol. 47, 687– 694 63 Sun, A. et al. (2011) Comparative cost analysis of algal oil production for biofuels. Energy 36, 5169–5179 64 Behrenfeld, M.J. et al. (2006) Climate-driven trends in contemporary ocean productivity. Nature 444, 752–755 65 Orcutt, D.M. and Patterson, G.W. (1975) Sterol, fatty acid and elemental composition of diatoms grown in chemically defined media. Comp. Biochem. Physiol. B 50, 579–583 123
Opinion 66 Knothe, G. (2005) Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process. Technol. 86, 1059– 1070 67 Durrett, T.P. et al. (2008) Plant triacylglycerols as feedstocks for the production of biofuels. Plant J. 54, 593–607 68 McCormick, R.L. et al. (2001) Impact of biodiesel source material and chemical structure on emissions of criteria pollutants from a heavyduty engine. Environ. Sci. Technol. 35, 1742–1747 69 Knothe, G. and Dunn, R.O. (2003) Dependence of oil stability index of fatty compounds on their structure and concentration and presence of metals. J. Am. Oil Chem. Soc. 80, 1021–1026 70 Tat, M.E. et al. (2007) Exhaust emissions from an engine fueled with biodiesel from high-oleic soybeans. J. Am. Oil Chem. Soc. 84, 865– 869 71 Buhr, T. et al. (2002) Ribozyme termination of RNA transcripts downregulate seed fatty acid genes in transgenic soybean. Plant J. 30, 155– 163 72 Renaud, S.M. et al. (2002) Effect of temperature on growth, chemical composition and fatty acid composition of tropical Australian microalgae grown in batch cultures. Aquaculture 211, 195–214
124
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73 Opute, F.I. (1974) Lipid and fatty-acid composition of diatoms. J. Exp. Bot. 25, 823–835 74 Orcutt, D.M. and Patterson, G.W. (1974) Effect of light intensity upon lipid composition of Nitzschia closterium (Cylindrotheca fusiformis). Lipids 9, 1000–1003 75 Ryther, J.H. (1959) Potential productivity of the sea. Science 130, 602– 608 76 Benemann, J.R. et al. (1982) Microalgae as a source of liquid fuels. In Proceedings of the June 1982 SERI Biomass Program Principal Investigators’ Review Meeting, Aquatic Species Program Reports 77 Benemann, J.R. and Oswald, W.J. (1996) Systems and economic analysis of microalgae ponds for conversion of CO2 to biomass. Final report. No. DOE/PC/93204–T5. California Univ., Berkeley, CA (United States). Dept. of Civil Engineering 78 Weissman, J.C. and Goebel R.P. (1987) Design and analysis of microalgal open pond systems for the purpose of producing fuels: a subcontract report. No. SERI/STR-231-2840. Golden, CO (USA), Solar Energy Research Inst. 79 Falkowski, P.G. and Raven, J.A. (2007) Aquatic Photosynthesis, Princeton University Press