Diatoms: a fossil fuel of the futureOrly 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, USA2 Department of Agriculture, Food & Resource Economics, Rutgers University, New Brunswick, NJ 08901, USA3 Department of Earth and Planetary Sciences, Rutgers University, Piscataway, NJ 0885, USA

Opinion

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

Long-term global climate change, caused by burningpetroleum and other fossil fuels, has motivated an urgentneed to develop renewable, carbon-neutral, economicallyviable alternatives to displace petroleum using existinginfrastructure. Algal feedstocks are promising candidatereplacements as a ‘drop-in’ fuel. Here, we focus on aspecific algal taxon, diatoms, to become the fossil fuelof the future. We summarize past attempts to obtainsuitable diatom strains, propose future directions for theirgenetic manipulation, and offer biotechnological path-ways to improve yield. We calculate that the yieldsobtained by using diatoms as a production platform aretheoretically sufficient to satisfy the total oil consumptionof the US, using between 3 and 5% of its land area.

The need for carbon-neutral fuelsThe first major oil well, drilled in 1859 by Edwin Drake,supplied cheap fuel for kerosene lamps, and led to a dramaticreduction in the demand for whale blubber. Although the useof kerosene as a fuel for lighting can be claimed as savingwhales from being hunted to extinction, there were otherunintended consequences to follow. The subsequent inven-tion of internal combustion engines provided a huge demandfor gasoline, which previously had been a worthless bypro-duct of kerosene distillation. By the early decades of the 20thcentury, the oil industry had become the engine of economicgrowth in industrializing nations. A legacy of Drake’s oil wellis that over 150 years later, �96% of all transportationprocesses in the world is still based on petroleum [1]. Theproven global reserves are projected to be able to meet thepredicted demand for several decades (http://energy.gov/fe/services/petroleum-reserves). However, an unintended con-sequence of the rapid combustion of fossil fuels is the rise ingreenhouse gas emissions. Since the beginning of the Indus-trial Revolution, >350 billion metric tons of carbon havebeen emitted into the atmosphere, with a commitment risein atmospheric CO2 of �43% over the past 150 years (http://petrolog.typepad.com/climate_change/2010/01/cumulative-emissions-of-co2.html, http://www.esrl.noaa.gov/gmd/ccgg/

0167-7799/$ – see front matter

� 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.01.004

Corresponding authors: Levitan, O. (levitan@marine.rutgers.edu); Dinamarca, J.(jotadin@gmail.com).Keywords: biofuel; diatoms; lipids; biomass; productivity; Energy Return (On)Investment.

trends/). Approximately 36% of the increase in atmosphericCO2 is a direct result of the combustion of petroleum fortransportation purposes [2]. The potential environmentalconsequences [2] have led to an urgent need to developrenewable, carbon-neutral, fuels that can directly displacepetroleum. Alga-based fuels potentially meet these criteria.However to date, their market penetration has been negli-gible. We review the potential for a specific algal taxon,diatoms, to become the biofuel of the future.

Diatoms are major sources of fossil fuelsDiatoms (see Glossary) are unicellular eukaryotic algaethat entered the fossil record �150 million years ago [3].They are secondary symbionts, distinguished from mostother algal forms by possessing a siliceous shell, or frus-tule. They rose to ecological prominence �34 million yearsago in the Oligocene, with the opening of the Drake Pas-sage and subsequent global cooling [4]. Their ecologicalsuccess introduced a major source of organic carbon formarine food webs, leading to the formation of massivefisheries – including whales [5]. A significant portion ofdiatom blooms sink along continental margins and shallowseas. Over geological time, a small fraction of this sinkingflux was converted to petroleum.

In the past 70 years, geochemists proved that algallipids are the major feedstock for petroleum. The sourceof the algae can often be traced from analysis of lipids thatact as biomarkers, and are stable over several million yearsin the petroleum reservoir [6]. The main biomarkers for

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. 3 117

O

O

HO

O

R2

R1

O

R3

OOH

OH

OH

OHR2

R3

R1

OH

OH O

O

O

HO –– CH3

HO –– CH3

HO –– CH3+ +

Triacylglycerol Methanol(alcohol)

Glycerol Met hylesters

0.0 0. 5 1. 00

5.0 × 10–07

1.0 × 10–06

1.5 × 10–06

Growth rate (d–1 )

TAG

acc

umul

a�on

(nm

ol T

AG d

–1)

Exponen�al phase Sta�ona ry phase

Exponen�al phase

Sta�ona ry phase

C25 C30

C28

C29

(A)

(B)

(C)

(D)

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.

Opinion Trends in Biotechnology March 2014, Vol. 32, No. 3

diatoms in petroleum are the ratio of steranes containing28 and 29 carbon atoms [7,8], 24-norcholestanes [9,10], andhighly branched isoprenoid (HBI) alkenes (Figure 1A) [11].These biomarkers are found in many of the highest qualityoil fields around the world.

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Targeting diatom lipids for biofuel productionTo date, biofuels have been clustered into four generationsof innovation. The first and second generations are basedon higher plant oils, which can satisfy only a small fractionof the existing demand for transportation fuels, without

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 cold-

temperature 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 Predominant fatty acids (>10%) Other fatty acids (1–10%) Refs

Phaeodactylum tricornutum 16:0, 16:1, 20:5 14:0, 16:2, 16:3, 18:1, 18:2 [65]

Thallasiosira psuedonana 14:0, 16:12, 20:5 16:0, 16:2, 16:3, 18:1, 18:2 [65]

Chaetoceros sp. 14:0, 16:1 16:0, 18:1, 16:3, 20:4, 20:5, 22:6 [72]

Navicula inserta 16:0, 16:1, 20:5 14:0, 16:2, 16:3, 18:2 [73]

Navicula muralis 16:0, 16:1 14:0, 16:2, 16:3, 18:2, 20:4, 20:5 [73]

Navicula pelliculosa 16:1, 20:5 14:0, 16:0, 16:2, 16:3, 18:1, 18:2 [65]

Nitzschia closterium 16:0, 16:1, 20:5 14:0, 16:3, 18:1, 18:2 [74]

Nitzschia palea 16:0, 16:1, 20:5 14:0, 16:2, 16:3, 18:2, 20:4 [73]

Nitzschia closterium 16:1, 20:5 14:0, 16:0, 16:2, 16:3, 18:1, 18:2 [65]

Nitzschia longissima 16:0, 16:1 14:0, 16:3, 18:1, 18:2, 20:4, 20:5 [65]

Nitzschia ovalis 16:0, 16:1 14:0, 16:2, 16:3, 18:1, 18:2, 20:4, 20:5 [65]

Nitzschia frustulum 16:0, 16:1 14:0, 16:2, 16:3, 18:1, 20:5 [65]

Cyclotella cryptica 16:0, 16:1, 16:3, 20:5 14:0, 22:6 [44]

Amphora exigua 16:0, 16:1 14:0, 16:2, 16:3, 18:2, 18:1, 20:5 [65]

Amphora sp. 16:0, 16:1, 20:5 14:0, 16:2, 16:3, 18:1 [65]

Biddulphia aurica 14:0, 16:1, 20:5 16:0, 16:2, 16:3 [65]

Fragilaria sp. 14:0, 16:1, 20:5 16:0, 16:2, 16:3, 18:2 [65]

Opinion Trends in Biotechnology March 2014, Vol. 32, No. 3

competing with crops for food and other resources [12,13].The third and fourth generation biofuels are based onalgae, and have received a great deal of attention in thepast �50 years.

Diatoms are extremely successful in the contemporaryoceans. They often outcompete other algae in mixed cul-tures [14,15] and are relatively resistant to pathogens[16,17]. Their major carbon storage product is lipids, espe-cially triacylglycerides (TAGs) [18], and under normalgrowth conditions, between 15 and 25% of their biomassis composed of fatty acids (Box 1). In the early 1980s, theAquatic Species Program, under the auspices of the USDepartment of Energy (ASP, http://www.nrel.gov/biomass/pdfs/24190.pdf) screened 3000 algal strains for their poten-tial to produce lipids. Of these, 50 strains were identified asworthy of consideration for commercial production; �60%of these were diatoms [19–21]. In fact, in a survey of 30species of algae, it was found that diatoms could reach anaverage of 25% lipids/dry weight during exponentialgrowth; 8% higher than green algae [22]. A comparisonof the strengths and weaknesses of green algae and dia-toms for biofuel production was reviewed by Hildebrandet al. [23]. Based on their lipid profile [20] and physiologicalcharacteristics, diatoms clearly are an underexploited,underappreciated biotechnological target for biofuel pro-duction (Box 1).

The scaling problem in displacing petroleumThe physical and chemical properties of algal biodiesel aresimilar to petroleum-based diesel fuels (the latter beingderived from the former), and thus require little or nomodifications for use in conventional engines [24–26].Yet, to be economically competitive with fossil petroleum,there are major hurdles to be overcome, starting withidentifying the best strains through optimizing cultivation,harvesting, extracting, and refining. Engineering solutionsto these problems are required to lower the production costper barrel of algal biofuel from the estimated $300 or moreto compete with the petroleum at �$100 per barrel cur-rently on the world market. In the USA alone, petroleumconsumption is �20 million barrels of oil per day (http://www.eia.gov/). Is it feasible to displace the demand with analga-based fuel?

From an economic perspective, the difference betweenextraction and refining petroleum and the production ofbiodiesel from algal mass cultures is in efficiency. At most,0.1% of algal biomass enters the sediments of shallow seasand continental margins [27]. Of this, between 0.001 and0.0001% becomes incorporated into a petroleum reservoirover geological time [28,29]. Although this deposition rate isextremely low, over geological time huge reservoirs of pet-roleum have formed. Each year, humans extract �1 millionyears worth of accumulated oil. To match that source

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Box 2. Biomass productivity of diatoms grown in outdoor systems

Figure I summarizes experimentally measured and calculated produc-

tivity 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 m�2 d�1

(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:

EROI ¼energyOUTþ

Pj 2 J

ajoj

energyINþP

k 2 KbkIk

, where: energyIN is direct energy flows

including electricity and fuel consumed during production; energy-

OUT is biofuel produced; K is non-energy inputs where k2K; J is non-

energy 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

Weismann and Goebel 1987

Benemann et al., 1982

Huesemann et al., 2009

Sheehan et al., 1998

Goldman and Ryther, 1975

Ryther, 1959 (high irradiance)

Ryther, 1959 (mid irradiance)

0 10 20 30 40

Biomass produc�vity from systemdesign costs and produc�vi�es foroutdoor pond

Measured biomass produc�vity ofdiatoms growing in outdoor ponds

Calculated produc�vity of marinephytoplakton

Biomass produc�vity (g/m2/d 1)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].

Opinion Trends in Biotechnology March 2014, Vol. 32, No. 3

requires a highly efficient production system; nevertheless,the task is far from impossible. For heuristic purposes wecan make the calculations with diatoms as the feedstock.

Gallagher [30] calculated that for algae to be economic-ally viable, the system productivity should be in the rangeof 100 mt/ha/year. The published productivity data fordiatoms ranges between 8 and 39 g/m2/day (Box 2), whichcorresponds to 29–142 mt/ha/year, or 0.6–2.7 g C/m2/day indiatoms. Based on these values, we calculate that diatomproduction in outdoor ponds can meet the defined baselinetargets [30] (Box 2, Table 1). Moreover, taking into con-sideration the mid and high productivity values of diatoms(Table 2) and a lipid content of 35% by weight (see Table S1in the supplementary material online) [30], we calculatethat the annual yield of diatom-based biodiesel canapproach 9000–15 000 gallons per hectare (Table 1). Willthis be enough to supply the USA consumption demands?

According to theoretical biodiesel yield calculations, itappears that 100% of the present demand for oil in the USAcould be met using only 3% of USA land area. Is thatphysically possible? The limiting factor that physicallylimits the production of alga-based biofuels is light. Givenan average of �3�1025 photosynthetically active quanta/m2/day incident within 358 latitude on either side of theequator, and assuming a modest photosynthetic energyconversion efficiency of 3%, with 35% of the photosyntheticproduct directed toward lipids, the system would produce

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1 g C/m2/day. Given that the daily rate of petroleum con-sumption in the USA is �24�1011 g C/day, the productionof diatom lipids would require �5% of the land area of theUSA. These calculations clearly demonstrate the potentialefficiency of diatoms as a source of carbon-neutral trans-portation fuels.

Boosting lipid production in diatoms usingenvironmental manipulationsThe production of diatoms in photobioreactors, as well asindoor and outdoor ponds, has been demonstrated for >50years [31–33], and large-scale culture of diatoms is used forfeeding shrimp and mollusks in commercial aquaculture[34–36], but diatoms have been largly neglected as a biofuelfeedstock. In the laboratory, many researchers havesearched for the ‘sweet spot’ of controlling the ‘carbon deci-sion tree’ for switching between biomass accumulation andlipid production in algae in general, and diatoms in parti-cular (Box 3). We summarize the third and fourth generationattempts to increase lipid accumulation in a variety ofmarine and freshwater diatoms from 1965 to 2013 (TableS1). In general, when grown under different conditions,diatoms can accumulate between 25% and 45% lipids ona dry weight basis, which is remarkably high [20,37].

Nitrogen starvation leads to accumulation of total lipidsand total fatty acids (TFAs) and increases the proportion ofTAGs (Table S1) [38–41]. However, nitrogen starvation

Table 2. Difference in dry weight and TAG yield for P. tricornutum during exponential and stationary growth phases

g/cell (•10S11) g/L (•10S1) Harvests per

year

g/l/year mt/ha/year

exp stat exp stat Exp stat exp stat exp stat

Biomass (dry weight) 5.7 4.4 0.57 4.4 120 45 6.84 19.80 13.6 39.6

TAG 0.05 0.37 0.005 0.37 120 45 0.06 1.65 0.12 3.3

Table 1. Biodiesel yield calculation and land area required for growth based on published productivity values reported for diatomsgrowing in outdoor pondsa

Biomass productivity

(mt/ha/yr)

Average lipid % Biodiesel yield

(gallons/ha/yr)

Overall area in acres

to reach 5 million

barrels per day

Percentage of the USA

land area needed to grow

5 million barrels per day

Low diatom productivity

(8 g/m2/yr)

29 35 3022 46 239 606 2.0

Medium diatom productivity(24 g/m2/yr)

86 35 8962 15 592 425 0.7

High diatom productivity

(39 g/m2/yr)

142 35 14 798 9443 300 0.4

aThe biodiesel yield was calculated based on a conversion ratio obtained from Gallagher [30].

Opinion Trends in Biotechnology March 2014, Vol. 32, No. 3

inevitability leads to reduced growth rates and may not bethe best third-generation approach to increase productiv-ity of diatoms [23,37].

An alternative approach to enhancing lipid accumula-tion is silicon starvation. Diatoms accumulate TAG undersilicon limitation without suffering from physiologicaldamage observed under nitrogen starvation. A study onnine diatoms showed that silicon starvation increasedtheir lipid content from 28 to 61%/dry weight, with anaverage of 45�10% (Table S1). In fact, silicon starvationoften leads to a higher accumulation of total lipids indiatoms than nitrogen starvation [18,21,23,41–44].

TAG accumulation in diatoms could also be enhancedwhen the cell cycle is arrested [45,46]. Lipids are primarilysynthesized in the G1 phase of the cell division cycle. InNitzschia palea, lipids accumulate in the presence ofan 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 carbox-

ylating 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.

in our group showed that arresting the cell cycle of Phaeo-dactylum tricornutum using a cyclin-dependent kinase 1and 2 inhibitor increases the cellular TAG content by �14-fold [47]. Thalassiosira pseudonana also exhibits rapidaccumulation of lipids in the presence of a microtubule-based inhibitor [23].

Diatoms as fourth-generation biofuelsAlthough third-generation strategies may prevail, geneticmanipulation of cells is also possible. The aim of the so-called fourth-generation biofuel is to co-opt basic biochem-ical pathways by using molecular genetic tools to generatephotoautotrophic algal strains with high lipid yield. Todate, there is abundant literature on genetic manipulationof algae that focuses on green algae in general, and onChlamydomonas reinhardtii in particular. However, it isunlikely that this freshwater green alga will be a commer-cial biofuel producer [48].

Although diatoms are diploid during normal growth,they do not have an easily controlled sexual recombinationphase. Therefore, using breeding approaches and/or clas-sical genetics to select for specific traits is not practical.However, genetic transformation of diatoms has beenreported since the 1990s [19,49–52]. Annotated genomesof P. tricornutum and T. pseudonana are published [53,54],and raw sequence data from the genomes of Fragilariopsiscylindrus and Pseudo-nitzschia multiseries are available(http://genome.jgi.doe.gov/). Together with new informa-tion from flux balance and transcriptomic analyses, trans-formation techniques [50,55,56] can be used to increasediatom lipid production efficiency.

Only a few studies describing genetic manipulation ofdiatoms to increase their biomass or lipid content have beenpublished to date (Table S1). Insertion of acetyl-CoA carbox-ylase gene (acc1) to Cyclotella cryptica led to increasedactivity of the enzyme [19,21], and overexpression of twoplant thioesterases (C14-TE and C12-TE) from Cinnamo-mum camphora increased the accumulation of short satu-rated chain length fatty acids (FAs) in P. tricornutum [57].Yet, neither transformation increased the total cellular lipid

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Opinion Trends in Biotechnology March 2014, Vol. 32, No. 3

content nor yielded any significant increase in secretion ofFA. Huesemann et al. [58] obtained a Cyclotella sp. strainwith higher photosynthetic saturation point, however, noimprovements in biomass productivities were observed. Theengineering of P. tricornutum to uptake glucose by expres-sing the human glucose transporter glut1 led to a tenfoldincrease in cell density under heterotrophic growth condi-tions [59]. This may be promising from the standpoint ofincreasing biomass, however heterotrophic growth of algaefor fuels defeats the purpose of obtaining a carbon-neutralfuel source. Recently, T. pseudonana with a multifunctionallipase/phospholipase/acyltransferase knockdown wasshown to increase lipid yields without affecting growth [52].

Diatom-based fourth-generation biofuel is clearly in itsinfancy. Nonetheless, targeting nitrogen uptake, the glu-tamine synthetase/glutamine oxoglutarate aminotransfer-ase pathway, and TAG biosynthesis appear to be promisingdirections for genetic manipulations [39,60,61]. We suggestthat testing the alteration of those branch points whilefinding the best growth conditions for the obtained strainswill bring us closer to mass production of biofuel. Finally,increased resistance to biotic stresses (competitors, para-sites, and pathogens) by introduction of new genes (e.g.,herbicides) could improve the sustainability of the systemand further increase the Energy Return (On) Investment(EROI).

Optimizing the efficiency of lipid productionAn objective metric of a production system is based on theconcept of EROI, which is the ratio of the energy obtainedto the amount of energy invested. Although there are largeuncertainties at virtually all steps of the biofuel productionprocess, an EROI higher than the break-even value of 1requires an algal biomass productivity above �17 g/m/day,corresponding to �1.2 g C/m/day, in purely photosyntheticsystems [62]. Energy costs could be influenced by mana-ging nutrient supply, lipid extraction, and co-production ofhigh-value compounds. To date, there are no EROI evalua-tions of any diatom feedstock, either in the laboratory orthe field. Yet, manipulating the productivity of the produc-tion line will result in a higher EROI.

In addition, the cost estimation of biofuel productiondepends on the number of harvests per year [63]. A com-parison of exponential and stationary phase P. tricornutumcultures, in terms of their culture densities, dry weight,and lipid profile [38,39] (Figure 1), suggests that harvest-ing during stationary phase (on Day 8) versus exponentialphase (Day 3) results in a threefold increase in biomass anda 38-fold increase in TAG (Table 2). Reducing the numberof harvests can also increase the EROI by reducing theenergy investment in two of the most expensive compo-nents of the production system: cultivation and harvesting.In addition to productivity per se, our analyses also showthat the diversity of FA increased during stationary phase,changing from mostly 16:0 and 16:1 during exponentialphase to include significant amounts of 14:0, 18:0, 18:2, and20:5 during stationary phase.

Concluding remarks and future perspectivesA major portion of current high-quality petroleum isderived from fossilized diatoms. The rise of diatoms to

122

ecological prominence over the past �34 million years isdue to their high photosynthetic energy conversion effi-ciency and rapid uptake and assimilation of nutrients. Itwould be worthwhile to take advantage of the naturalselection of these organisms that have evolved such highphotosynthetic energy conversation with lipids as theirprimary storage product. Growing diatoms as feedstockfor biofuel production could displace all petroleum con-sumption in the USA. However, it should be clear that thefuture of biofuels is based on a technological conundrum:the more one makes the biological product, the cheaper thefossil product will become. Consequently, the displacementof fossil fuels will require more than technologicaladvances and investment in infrastructure; it will requirestrong market incentives, such as ‘wedge taxes’ that fix thecosts of petroleum to match that of algal biofuels. Withoutpolicy intervention, the continued reliance on fossil fuelswill further distort global climate systems, leading to areduction in oceanic primary production [64] and in foodsupplies to higher trophic levels, including whales. Ironi-cally, although the exploitation of petroleum may havesaved whales 150 years ago, the increased use of oil as afuel may ultimately lead to the demise of these and otheranimals in the coming century.

AcknowledgmentsOur 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. SmithEndowment, and the Rutgers Energy Institute. We thank Benjamin VanMooy for analyzing the TAG amounts and composition of our Phaeodac-tylum tricornutum cultures. We thank Robert Kopp for comments aboutlife cycle assessments. Confocal microscopy to view lipid bodies was doneat Rutgers University SEBS Core Facility and, funded byNIH1S10RR025424 to N. E. Tumer, with the assistance of Michael D.Pierce.

Appendix A. Supplementary dataSupplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.tibtech.2014.01.004.

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