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Review
The place of diatoms in the biofuels industry
Biofuels (2012) 3(2), 221–240
Mark Hildebrand*, Aubrey K Davis, Sarah R Smith, Jesse C Traller & Raffaela Abbriano
In spite of attractive attributes, diatoms are underrepresented in research and literature related to the
development of microalgal biofuels. Diatoms are highly diverse and have substantial evolutionarily-based
differences in cellular organization and metabolic processes relative to chlorophytes. Diatoms have
tremendous ecological success, with typically higher productivity than other algal classes, which may relate
to cellular factors discussed in this review. Diatoms can accumulate lipid equivalently or to a greater extent
than other algal classes, and can rapidly induce triacylglycerol under Si limitation, avoiding the detrimental
effects on photosynthesis, gene expression and protein content associated with N limitation. Diatoms have
been grown on production scales for aquaculture for decades, produce value-added products and are
amenable to omic and genetic manipulation approaches. In this article, we highlight beneficial attributes
and address potential concerns of diatoms as biofuels research and production organisms, and encourage a
greater emphasis on their development in the biofuels arena.
A frequently stated reason for choosing unicellular
microalgae as biofuel-production organisms is that they
are more efficient than vascular plants at turning light
and nutrients into valuable products. The economics
of algal biofuels is critically dependent on productivity, and one can apply the same criterion and ask the
question: among the microalgae, are certain classes
more productive? Before answering that question, we
need to acknowledge that within a given class of algae,
there will be differences in measured productivity values when comparing individual species. These differences can stem from intrinsic factors related to their
cell biology or from growth conditions at the time of
sampling, which can involve both environmental and
ecological variables. Comparing productivity in controlled culture conditions has value, because it reflects
what is possible, but it also encompasses biases due to
the fact that conditions are never exactly the same in
two culture systems. Indications are that algal biofuels
will be developed on a regional basis in a wide variety of
environments [1] . Another consideration is the utility of
comparing maximum productivity values. In a production system operating for a good portion of the year,
it would be far more desirable to have strains that are
consistently productive under a variety of environmental conditions than strains that have higher productivity
under optimal conditions for a shorter period of time. A
final consideration in productivity comparisons is that
if the number of studies is relatively small, which is
usually the case, poor statistical comparisons result. An
alternative to measuring productivity values in culture
is to evaluate an algal class’ productivity on the basis of
its abundance in nature in a variety of environments.
This approach may directly relate to biofuel production
systems that would be deployed in different environments and provide robust statistics, since the sample
size is large.
Diatoms (Figure 1) are among the most productive and
environmentally flexible eukaryotic microalgae on the
planet. They are estimated to be responsible for 20%
of global carbon fixation and have become the dominant primary producers in the ocean [2,3] . In addition
Marine Biology Research Division, Scripps Institution of Oceanography, University of California, 9500 Gilman Dr., La Jolla, CA 92093-0202, USA
*Author for correspondence: Tel.: +1 858 822 0167; E-mail: [email protected]
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10.4155/BFS.11.157 © M Hildebrand
ISSN 1759-7269
221
Review Hildebrand, Davis, Smith, Traller & Abbriano
A
C
B
1 µm
10 µm
F
E
2 µm
D
20 µm
G
10 µm
100 µm
H
4 µm
20 µm
Figure 1. Scanning electron micrographs of different diatom species. (A) Thalassiosira pseudonana, (B) Cocconeis sp.,
(C) Lampriscus sp., (D) Gyrosigma balticum, (E) Cyclotella cryptica, (F) Nitzschia sp., (G) Thalassiosira weissflogii and (H) Achnanthes sp.
to being highly abundant, they are also highly diverse,
with estimates of over 100,000 species [4] . Their most
distinctive cellular feature is their cell wall made of
nanostructured silica (Figure 1) , which is reproduced
with fidelity through generations by genetically controlled assembly processes. Typical diatoms range in size
from a few to a few hundred microns, with the majority
of species between 10 and 50 µm.
Diatoms have characteristics, both conceptual and
proven, which make them amenable to large-scale biofuel cultivation. In 1998, the Aquatic Species Program
recommended a list of 50 microalgal strains, selected
from a pool of over 3000 candidates, which held the
most promise as biofuel-production organisms [201] .
Sixty percent of the selected strains were diatoms, which
were chosen based on criteria such as high growth rates
and lipid yields, tolerance of harsh environmental conditions, and performance in large-scale cultures [201] .
Diatoms are excellent lipid accumulators, and we frequently see cells similar to the ones shown in Figure 2
where a substantial portion of the cells’ volume is occupied by lipid droplets that accumulate rapidly, especially
under Si limitation.
There is a puzzling underrepreKey terms
sentation of diatoms in the microProductivity: The ability of a species to
algal biofuels arena. Based on our
grow rapidly to high density and
survey of the literature and research
accumulate fuel precursor molecules.
experience, this is not because they
Diatoms: Unicellular microalgae of the
are inferior to other classes of algae.
heterokont class, characterized by
silica-based cell walls and high
The purpose of this article is to
productivity.
highlight attributes of diatoms that
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Biofuels (2012) 3(2)
make them highly desirable as organisms for biofuel
research and production, and to address potential concerns and misconceptions about diatoms that we have
encountered in the literature and during dialog with
colleagues over the past several years.
Evolutionary aspects of diatoms
Diatoms are members of the Stramenopile or heterokont class of algae (Figure 3) , which are highly different
from, and have a more complex evolutionary history
than, green algae and vascular plants [5] . Common to
all photosynthetic eukaryotes was a primary endosymbiosis event where a heterotrophic eukaryote engulfed
or was invaded by a cyanobacterium. Most of the cyano­
bacterial genes were transferred to the nucleus as the
cyanobacterium evolved into the chloroplast. The progenitor plant cell resulting from primary endosymbiosis gave rise to the glaucophytes, red algae, green algae
and plants. A secondary endosymbiosis later occurred
between a different heterotrophic eukaryote and a red
alga. The red algal endosymbiont was the progenitor
of the plastids found in Stramenopiles, a group that
includes diatoms, brown macroalgae and plant parasites
(Figure 3) . In addition to diatoms, prospective biofuel
production organisms such as Nannochloropsis are in
the Stramenopiles.
The evolutionary story is a bit more complex for
diatoms; evidence suggests that at some stage in the
development of the progenitor plant cell, a Chlamydial
invasion occurred [6] . There is also evidence that
green algal nuclear genes are present in the secondary
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The place of diatoms in the biofuels industry Review
A
B
C
5 µm
5 µm
D
5 µm
E
5 µm
5 µm
Figure 2. Fluorescence and light micrographs of lipid droplets in diatoms. (A) Nitzschia curvilineata, (B) Cyclotella cryptica,
(C) C. cryptica, (D) Nitzschia alba and (E) Nitzschia filiformis. Green fluorescence is BODIPY staining of neutral lipids, red is chlorophyll
autofluorescence. (B) is a differential interference contrast image with no fluorescence.
endosymbiont [7] , suggesting an endosymbiotic episode
with a green alga. Diatom genomes also contain abundant numbers (up to 5%) of genes derived from bacteria
of various classes, with more than half of these genes
being shared between two evolutionarily diverse diatoms,
Thalassiosira pseudonana and Phaeodactylum tricornutum
[8] . Thus diatom genomes, and their resultant metabolomes, are a complex mixture of components derived
from highly divergent sources [8,9] . This is reflected in a
unique combination of metabolic processes in diatoms,
which combine both plant- and animal-like characteristics [10] . Fundamental aspects of diatom metabolism
differ substantially from other classes of algae [11] , and
caution should be taken when making generalizations
about algae based on studies on one organism.
The four major groups of diatoms (based on the features of their silica cell walls) are the radial centrics, the
bipolar and multipolar centrics, the araphid pennates,
and the raphid pennates; each group arose and diversified sequentially under decreasing CO2 levels during the
Mesozoic era [5] . Thus, their carbon concentrating and
fixation systems evolved under substantially different
conditions; for example, CO2 levels were, at one point,
eight-times higher than present [12] . It is reasonable to
expect that changing environmental CO2 levels were
drivers (at least in part) of diatom diversification and
that differences in the physiology of the major groups
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may reflect this adaptation. Diatoms also adapted to
grow and dominate in low-nutrient open ocean waters
resulting from changing global geochemical conditions
[5] . Conversely, diatoms may have affected environmental conditions themselves; a rapid rise in diatom abundance during the Eocene may have resulted in a massive
drawdown of atmospheric CO2 that facilitated global
cooling [13] .
The fossil record indicates that over the Mesozoic era
larger eukaryotic phytoplankton of the red algal lineage,
which includes dinoflagellates, coccolithophorids and
diatoms, displaced a large portion of other algae in the
ocean, which were predominantly cyanobacteria and
very small green algae [14] . This suggests fundamental
productivity advantages to the red algal line [14] , which
will be discussed later. Enhanced sinking rates of the
larger phytoplankton increased the burial of organic carbon in the continental margins and shallow seas, which
generated most of our known petroleum reserves [15,16] ,
and reduced atmospheric CO2 levels. While some early
reports suggested that diatoms were the major source of
carbon for fossil fuels, dinoflagellates and coccolithophorids were the dominant types of phytoplankton
during the major carbon export period [14] . Currently,
diatoms are responsible for a large fraction of the organic
carbon buried on continental margins [17] and are major
contributors to nascent petroleum reserves.
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Review Hildebrand, Davis, Smith, Traller & Abbriano
Class
Red algae
Diatom productivity
Currently, in the oceans, diatoms are
one of the most productive classes
of microalgae. They are responsible
for a substantial portion (40%) of
marine primary productivity [3] and
most of the CO2 export (drawdown
and sinking) from the atmosphere,
of the order of 50% [18] . The intrinsically high productivity of diatoms
not only makes them major players in the global carbon cycle, but
also suggests their utility as biofuel
production organisms.
Glaucophytes
ƒƒ Natural productivity
Group
Land plants
Charophytes
Streptophytes
Chlorophytes
Trebouxiophytes
Ulvophytes
Prasinophytes
Mesostigma
Green algae
Floridiophytes
Banglophytes
Cyanidiophytes
Glaucophytes
Apicomplexa
Colopella
Dinoflagellates
Oxyhrris
Perkinsus
Ciliates
Colponema
Ellobiopsids
Alveolates
Diatoms
Bolidophytes
Raphidiophytes
Eustigmatophytes
Chrysophytes
Phaeophytes
Pelagophytes
Blastocystis
Actinophryids
Labyrinthulids
Thraustrochytrids
Oomycetes
Opalinids
Bicosoecids
Stramenopiles
Haptophytes
Haptophytes
Cryptomonads
Cryptomonads
Figure 3. Unrooted consensus phylogeny of photosynthetic eukaryotes and derived taxa.
Branch lengths do not correspond to phylogenetic distances. Red arrow locates diatoms and
green arrow locates chlorophytes.
Reproduced with permission from [176].
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Biofuels (2012) 3(2)
Diatoms are efficient at nutrient
utilization and are entirely responsible for the initial nitrate uptake
in the equatorial upwelling zone of
the eastern Pacific Ocean [18] . Other
eukaryotic phytoplankton obtain
their N through the action of grazers, which allows N from diatoms to
be recycled [18] . One of the contributing factors to diatom dominance
is that they possess a large nutrient
storage vacuole, which dinoflagellates and coccolithophorids lack
[19–21] . Vacuolization has important
consequences in terms of productivity. Nutrient utilization efficiency is
generally related to surface to volume ratio, and smaller cells tend
to be more efficient for this reason.
One way to compensate for a larger
surface area is to store nutrients
inside the cell in a vacuole, which in
essence reduces the cytoplasmic volume and increases the surface area
from which nutrients can be utilized
by the cytoplasm [19] . As long as
extracellular nutrients are present at
levels that minimize diffusive limitation to the cell surface, the gains
in productivity in vacuolated cells
are quite substantial [19] . Thus, in
nutrient-replete conditions, diatoms
will hoard excess nutrients, storing
enough nitrate to enable several cell
divisions, and preventing the growth
of other phytoplankton [15] . In a survey of in situ growth rates of marine
phytoplankton, maximum doubling
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The place of diatoms in the biofuels industry Review
rates between 2 and 4 per day were repeatedly measured
for both pennate and centric diatoms, which were higher
than those for other algae with corresponding sizes [22] .
Diatoms can even dominate under nutrient-limiting
conditions; in one example a diatom species was shown
to outcompete non-N-fixing cyanobacteria under low
nitrate concentrations in a eutrophic lake [23] . Diatoms
consistently outcompete other eukaryotic phytoplankton under conditions of mixing and high turbulence, in
which pulses of nutrients occur between short periods of
quiescence [24,25] . Furthermore, the relative dominance
of diatoms in the oceans relates to geologic time periods
of generally greater ocean turbulence [26–29] .
Diatoms also have a greater carbon fixing ability than
other co-existing groups of microalgae, as shown from
calculations of productivity per unit of crop carbon
[30] . This has not only been repeatedly demonstrated
in laboratory cultures under optimal conditions [31–33] ,
but in environmental samples from different locations
[30] . In a comparative study, diatoms grew faster under
low light conditions than competing algae [34] . Higher
photosynthetic efficiency for diatoms not only relates to
other Stramenopiles, but also in comparison to green
algae (molecular mechanisms will be discussed below).
A comparison between P. tricornutum and Chlorella
vulgaris indicated that under nonfluctuating light conditions, efficiencies of converting light into biomass
were equivalent, but under fluctuating light conditions,
the diatom was nearly twice as efficient as C. vulgaris
[35] . Efficient carbon fixation ability along with nutrient utilization could increase the appeal of diatoms for
co-processes such as CO2 abatement and waste water
remediation.
ƒƒ The relevance of natural productivity to biofuels
production
Several of the concepts presented in the previous paragraphs are of relevance to biofuels production. Diatoms
naturally bloom under nutrient-replete conditions,
meaning maximal growth rate to highest density is
obtained, which are ideal characteristics for a biofuel
production system. Diatom blooms also tend to crash
precipitously, which is a tendency of all bloom species.
Crashes can result from a number of factors including
nutrient exhaustion, grazing, sedimentation, or viral
lysis and remains a fundamentally poorly understood
process in all microalgae [36,37] . Environmental studies have shown that once chlorophyll levels begin to
decrease (signaling the beginning of the end of the
bloom), triacylglycerol (TAG) content increases [38,39] .
Translating that into a biofuels production scenario,
TAG accumulation precedes cell death and thus crashing would not necessarily be detrimental to production.
Diatoms also grow best under highly mixed conditions,
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which are desirable for large-scale cultivation. Their
large contribution to carbon export in the oceans relates
to their relatively rapid sinking rates, which could translate into enhanced harvesting ability in a production
system (see below). Their ability to sequester nutrients
could be advantageous for crop management; supplying
a nutrient pulse in which most of a particular nutrient is rapidly taken up and removed from the culture
medium could reduce nutrient availability for contaminating organisms. Vacuole size and nutrient storage
capacity should affect different aspects of production
schemes. Chlorophytes, including Chlorella species,
Chlamydomonas reinhardtii and Dunaliella salina, have
small vacuoles (3–14% of their protoplast volume) compared with diatoms (45–90% of their protoplast volume), and should have a greater dependence on external
nutrient conditions including consistency of supply [19] .
In terms of lipid productivity, cells with larger vacuoles
may take longer than cells with smaller ones to become
nutrient limited, due to the enhanced nutrient storage
capability.
ƒƒ Diatom productivity in large-scale culture
systems
Diatoms have been cultivated in large-scale outdoor
systems for purposes other than biofuel production,
such as aquaculture, for decades [40] . Widely ranging
values for algal biomass productivity are reported in
the literature and are due in large part to differences
in cultivation parameters. Such parameters include,
but are not limited to, light source and intensity, depth
of culture, mixing rate, temperature, nutrients and
supplementary carbon additions in the form of sugars
or CO2. In an attempt to limit some of the variability
for the purpose of comparison, only values obtained
from microalgae grown in outdoor ponds, raceways or
open photobioreactors illuminated with natural light
are included here. Diatom biomass productivity values range from 5 to 25 g m-2 day-1 [30,41–43] , and are in
line with values obtained for other types of microalgae
including the Eustigmatophyte Nannochloropsis salina
[44] , the cyanobacterium Spirulinaplatensis [45] and the
Chlorophyte Chlorella sp. [46] , whose biomass productivity values range from 10–25 g m-2 day-1. Cyclotella
cryptica, a model diatom species from the Aquatic
Species Program, was shown to have consistent productivity levels of 20 g m-2 day-1 (erroneously listed as
12 g m-2 day-1 in the text of this paper [41]).
What cellular factors contribute to diatom
productivity?
As discussed previously, the evolution of diatoms is
more complex and unique than that of green algae and
terrestrial plants. As a result, there are fundamental
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Review Hildebrand, Davis, Smith, Traller & Abbriano
differences in the biology (i.e. metabolism, physiology and subcellular organization) of the different algal
classes [11] . Acknowledging diversity within a class, it
is still reasonable to expect there will be advantages or
disadvantages to certain classes in a large-scale biomass
production setting. It is useful to outline some of the
variation between classes so as to better understand the
biology of the organisms that we propose to cultivate
for fuel. Since chlorophytes are commonly considered as
algal biofuels production strains and are fundamentally
different than diatoms, we will focus on comparing these
two classes. We acknowledge that there are other classes
of photosynthetic organisms (Nannochloropsis, cyanobacteria) being considered for large-scale cultivation
that are not included in this discussion.
ƒƒ Differences in photosynthesis & carbon fixation
comparing diatoms & green algae
Dissipation of excess light in photosynthesis either occurs
at the level of photosystem II (PSII) and especially lightharvesting complex II (LHCII) via thermal dissipation
or adjustment of the absorption cross section; or by redirecting electrons into pathways other than CO2 fixation.
Diatoms possess an additional xanthophyll-based cycle
involved in energy dissipation relative to green algae and
higher plants [47] . In a comparative study, the diatom P.
tricornutum favored modulation at PSII/LHCII, with a
substantial increase in de-epoxidation of diadinoxanthin
to diatoxanthin, which reduced the flow of electrons
into secondary processes. This was more efficient than
C. vulgaris, where thermal dissipation at LHCII was less
[48] . Green algae undergo state transitions where reorganization of antenna proteins shifts primary activity
between PSII and PSI, especially under fluctuating light
conditions [49] . State transitions increase photosynthetic
efficiency, as has been demonstrated by mutants in this
process, which have reduced capacity to make ATP via
cyclic-photophosphorylation and have impaired growth
[49] . No evidence for state transitions or a reaction centerbased quenching mechanism has been found in diatoms
[50] , yet they are inherently more efficient than chlorophytes in balancing photosynthetic electron flow [48] .
Perhaps the lower efficiency of chlorophytes involves the
need for physical movements of antenna proteins over
500 nm [49] , as compared with localized quenching by
additional xanthophyll as occurs in diatoms. Diatom
thylakoid membranes are arranged in groups of three
and are not differentiated into grana and stroma lamellae [51,52] , which affects redox signaling. Diatoms lack
the a-carotene biosynthetic pathway, which means that
both photoprotective and light harvesting pigments
are synthesized from the same precursors, in contrast
to the chlorophytes [48] . In addition, in contrast to the
chlorophytes, diatom light-harvesting proteins are not
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Biofuels (2012) 3(2)
differentiated into major and minor complexes [53,54] , the
Calvin–Benson cycle is not controlled by light via the
redox state of thiol groups [55] and the oxidative pentose
phosphate cycle is apparently not present in the chloroplast [55] . It is reasonable to assume that these substantial
differences in photosynthesis, and carbon fixation and
flux, contribute to diatoms’ environmental success.
ƒƒ Carbon fixation efficiency & concentrating
mechanisms
Both diatoms and chlorophytes possess carbon concentrating mechanisms (CCMs), which effectively increase
CO2 concentration in the proximity of RuBisCO. It is
not clear whether there are advantages in CCM efficiency
intrinsic to either class; however, there are significant
differences in the ability of RuBisCO to discriminate
between CO2 and O2. The chlorophytes have Form1B
RuBisCO, with selectivity (Srel) values ranging from 54
to 83, whereas diatoms have Form1D RuBisCO, with
a much higher Srel of 106–114 [56] . CCMs can involve
two different mechanisms: biochemical (e.g., C4 metabolism), in which carbon is fixed by an enzyme other
than RuBisCO and the fixation product or a derivative
is decarboxylated in the vicinity of RuBisCO; or biophysical, which involves localized enhancement of CO2
via equilibrium shifts involving pH changes or active
transport of inorganic carbon across membranes [57] . The
latter involves carbonic anhydrase (CA) and bicarbonate
transporters. In diatoms, evidence for both C3 and C4
biochemical fixation exists, and the preference can apparently differ in relatively closely related species [58] , which
has led to confusion in attempts to make generalizations.
P. tricornutum and T. pseudonana encode 9 and 13 CAs,
respectively, but only P. tricornutum has b-CAs that are
targeted to the chloroplast [59] . It is as yet unclear whether
differences in CCMs relate to the different evolutionary
time periods under which different classes of diatoms
arose in relation to CO2 levels. The documented differences may either reflect an organization that maximizes
efficiency under a given CO2 condition or, alternatively,
that a variety of organizational schemes provide acceptable efficiency under current conditions. More research,
especially localization studies, is required to clarify the
situation. Variation in CCM mechanisms may not be
unexpected; according to a modeling study, the advantage of a CCM would be best suited for an intermediatesized algal cell, and there should be a high degree of
co-adaptation in different uptake systems [60] .
ƒƒ Compartmentation & carbon metabolism in
diatoms
Subcellular compartmentation enables incompatible metabolic processes to occur by physically separating them, and many biochemical processes (lipid
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The place of diatoms in the biofuels industry Review
metabolism, oxidative phosphorylation, photosynthesis) take place on the membranes of subcellular compartments, which also provide a greater area for these
processes to occur [61] . Stramenopiles have additional
intracellular compartmentation relative to chlorophytes
as a result of their secondary endosymbiotic origin. In
particular, the chloroplast is surrounded by two additional membranes, the chloroplast endoplasmic reticulum and the periplastidic membrane [62,63] . These two
membranes are closely associated, but there is a noticeable space between the periplastidic membrane and
the outer chloroplast membrane [64] . This periplastid
compartment appears to be functionally significant,
as several enzymes involved in myriad of cellular processes, including lipid transport and metabolism, have
been shown to be targeted there [65] . Though the specific
roles of this compartment are far from resolved, it is clear
that it plays a part in regulating metabolism and overall
cellular function.
Similar to green algae and terrestrial plants, diatom
genomes contain genes for the essential enzymes of the
conserved eukaryotic central carbon metabolic pathways
of carbon fixation, glycolysis/gluconeogenesis and the
citric acid cycle [8,10] . One difference in the glycolysis
pathway is that neither T. pseudonana nor P. tricornutum
encode a gene for a glucokinase [66] . Perhaps more surprising is the localization of the complete lower half of the
glycolysis pathway from triose phosphate isomerase to
pyruvate kinase to the mitochondria [66] . Diatoms apparently have the unique ability to convert glyceraldehyde-3phosphate into pyruvate, along with the attendant intermediates, entirely in the mitochondria. The role of this
partial pathway in the mitochondrion is unclear, though
it could be a strategy to channel substrates towards the
citric acid cycle. In terrestrial plants, an increased association between cytosolic glycolytic enzymes and the mitochondrial surface occurs during higher respiration, with
attendant-increased efficiency through substrate channeling [67] . A mitochondrial-localized glycolysis pathway
could also avoid the need for the malate-aspartate shuttle
to import the reducing equivalents needed to generate
ATP through oxidative phosphorylation.
Diatoms contain a complete urea cycle [10] , which is
not found in chlorophytes, and several metazoan-like
urea cycle components are localized to the diatom mitochondria. In metazoans, the ornithine-urea cycle is only
known for its essential role in the removal of fixed N; in
diatoms, however, it apparently serves as a distribution
and repackaging hub for inorganic carbon and N [68] .
Experimental manipulation of the cycle indicates that
it contributes significantly to the metabolic response of
diatoms to episodic N availability [68] .
Diatoms also differ from other classes of algae in terms
of carbohydrate storage. Instead of starch, diatoms store
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fixed carbon as a complex carbohydrate called chrysolaminarin, which contains b(1→3) and b(1→6) linked
glucose units in a ratio of 11:1 [69] . Chrysolaminarin
is soluble and is stored in the chrysolaminarin vacuole
(CV). The CV is quite large [70] , and recent imaging
work in our laboratory in collaboration with Robyn
Roth at Washington University (MO, USA) indicates
that it can comprise as much as 50% of the cell volume. Diatoms are not necessarily better at storing carbo­
hydrates than green algae. For example, C. reinhardtii
has quite an impressive ability to accumulate starch [71] .
However, it may be important to consider the relative
energetics involved in accessing a soluble carbohydrate
(chrysolaminarin) stored in a vacuole versus an insoluble
form (starch) stored in the chloroplast.
ƒƒ The silica cell wall
The most conspicuous feature of diatoms is their silicabased cell wall, also called the frustule (Figure 1) . The
frustule consists of two halves that overlap like a petri
dish, the epi- and hypo-theca. The ‘top’ and ‘bottom’
of the dish are made of structures called valves, and the
sides are most typically composed of a series of thin
siliceous bands that encircle the cell, overlap each other,
and provide the overlap between the theca, called the
girdle bands. During vegetative division, diatoms make
new valves in an intracellular compartment called the
silica deposition vesicle (SDV), and once complete, the
entire structure is exocytosed [72] . After the two daughter cells separate, individual cells can sequentially synthesize new girdle bands, which enable elongation of the
cell. The source of Si for diatoms is dissolved silicic acid,
Si(OH)4 [73] , which is taken up at low concentrations by
specific silicic acid transport proteins, and at high concentration by diffusion across the plasma membrane [74] .
The silica-based cell wall not only serves a protective
function, but can contribute to diatoms’ overall productivity. Silica is polymerized from soluble silicic acid
precursors in an energetically favorable autocatalytic
process when silicic acid concentrations are sufficiently
high (above 2 mM) and/or the pH is lowered [75] . In
diatoms there are organic components that speed up
this process; however, they are present in, at most, a
few percent relative to the silica that is formed. The
energetic needs for silica polymerization are thus much
lower than an organically based cell wall, and calculations done for silicified plant cell walls indicated that
silicification required only 3.7% of the energy compared
with a lignin-based wall, and 6.7% for a polysaccharide wall [76] . In addition to the energetic savings, there
should be savings in carbon, since a large percentage
of the carbonaceous cell wall components are replaced
with silica and the carbon can be used for other cellular
purposes. Another feature of the cell wall that may relate
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Review Hildebrand, Davis, Smith, Traller & Abbriano
Key term
to biofuels and specifically TAG
accumulation, is that very different
cell shapes can result, which may
impose constraints on the size and
number of lipid droplets that can be
accommodated in the cell (Figure 2) .
There may actually be species-specific geometrical
features that relate to optimal productivity.
The silica cell wall also apparently plays an important role in carbon concentrating mechanisms. Data
suggest that diatom biosilica is an effective pH buffer,
which increases carbonic anhydrase activity near the
cell surface to enable the conversion of bicarbonate
to CO2 [77] .
Lipid accumulation: The process of
triacylglycerol accumulation in cells
leading to the formation of lipid
droplets.
Attributes of diatoms for biofuel production
In addition to efficient photosynthesis and nutrient
utilization, diatoms possess several other attributes that
relate directly or indirectly to useful characteristics for
a biofuels production system.
ƒƒ Environmental & trophic flexibility of diatoms
It is envisioned that algal biofuels development will
have a regional character, and there will be a need to
develop strains optimally adapted to different environments [1] . Further, the development of different types
of culture strategies, ranging from outdoor open systems to enclosed photobioreactors to hybrid systems,
and different trophic strategies, from auto- to heteroto mixo-trophic, must be considered [78] . Thus algae
with diverse culturing characteristics are desirable.
On this note, diatoms are found in a wide variety of environments, including general environments
such as marine, freshwater and soil, but also specific
extreme or variable environments of salinity, temperature (ice or hot springs) or pH. The most intensively
studied diatoms are marine, and all of the species with
sequenced genomes fall into that category. This does
not preclude freshwater diatoms from being developed
for biofuels production, but considering the issue of
water supply, saline-tolerant species may be the most
appropriate for development. Diatoms isolated from
thalassic hypersaline marine environments include
both centric and pennate species that grow well at
salinities ranging from 0.5 to 15%, and three strains
of Pleurosigma strigosum cannot grow in salinities
less than 5% total salts, representing truly halophilic
diatoms [79] . Different strains of P. tricornutum were
shown to have near-linear growth rates (divisions
per day) in salinities ranging from 5 to 70 psu [80] .
Numerous species grow under acidic conditions (pH
3.3–4.4 [81]) and diatoms have been found to grow at
pHs consistently maintained between 1 and 2.5 [82] .
Conversely, a wide array of both pennate and centric
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Biofuels (2012) 3(2)
species have been found in alkaline lakes, such as
those in East Africa, where the pH can reach 10.6
[83] . Diatoms can grow at 60–65°C in hot springs [84]
and at 40°C in thermal muds, which are a 5 g l-1 colloidal aluminum silicate suspension mixed with thermomineral water, rich in sodium chloride and sulfates
[85] . Psychrophilic diatoms grow on polar ice and in
hypersaline brine channels at -1–2°C [86] , with photosynthetic activities optimized between 4 and 7°C [87] .
Diatoms can form biomasses in ice with chlorophyll
concentrations that are matched only in hypertrophic
waters or in extremely eutrophicated sediments [88] .
The Fragilariopsis genus may be the most prevalent
biomass producer among polar plankton [89,90] , serving
as the base of the entire polar food web. The range of
different environments where diatoms can dominate
sheds light on their diversity; in terms of biofuels production, growth in extreme conditions, such as salinity
or pH can be utilized to keep pond contaminants and
predators at low levels.
Diatoms exhibit trophic flexibility. The majority of
species are autotrophic, but mixotrophy and obligate
heterotrophy occur. C. cryptica, a candidate biofuels
production strain, is capable of slow growth (10% of
autotrophic) on glucose only in the dark, likely due to
its glucose transport mechanism being induced in the
absence of light [91] . The mixotrophs and obligate heterotrophs are generally able to use simple sugars as carbon sources, but some species are able to break down
complex carbohydrates. There are two forms of mixotrophs: those that can grow strictly auto- and heterotrophically, or those whose growth is only enhanced
in the presence of an extracellular carbon source [92] .
Several Nitzschia species can switch between strict
auto- and hetero-trophic growth [93] and have been
observed to burrow into solid medium containing agar,
presumably breaking it down as a carbon source. In
the natural environment, heterotrophic diatoms can be
found on macroalgae (kelp), from which they obtain
their carbon [94] . Nitzschia alba is an obligate heterotroph that has lost its chloroplasts, and can grow on
both simple and complex carbohydrates [94–96] , demonstrates growth rates in the field appreciably higher than
other heterotrophic protists, and doubles in 6 h when
cultured in the presence of glucose [95] . The ability of
diatoms to breakdown complex carbohydrates suggests
possible applications with different carbon feedstocks,
such as cellulose.
ƒƒ Lipid amount & composition
A primary target for biofuels production is the accumulation of lipid, either in the form of total lipid or
TAG, which provide carbon for conversion to a variety of types of fuel [1] . Diatoms are excellent lipid
future science group
The place of diatoms in the biofuels industry Review
accumulators, which partly may be due to extra buoyancy needed to counteract the effect of their relatively
heavy silica cell walls. It is vitally important when
comparing lipid content numbers to appreciate the
variability in conditions in different studies; strict
comparisons cannot generally be made. However, by
comparing data from a sufficiently large number of
studies some general conclusions can be formed.
In terms of total lipids, Shifrin and Chisholm [97]
reported values of 12–45% of dry cell weight (DCW)
for diatoms under conditions of N limitation. A comparative ana­lysis of several species in different algal
classes by Hu et al. [98] showed that on a DCW basis,
chlorophytes had an average total lipid content of
25.5% under normal growth and 45.7% under nutrient stress. Regarding diatoms, lipid content was 22.7%
DCW during normal growth and 44.6% during stress
as indicated in the text, but this value was mistakenly
listed as 37.8% in their Figure 1, as the line depicting the average is clearly above 40% [98] . A literature
survey by Griffiths and Harrison [99] of 55 microalgal
species in various classes showed that diatoms as a class
performed above average at 27.4% (19% better than
average) for total lipid content under nutrient-replete
conditions and 36.1% (13% better than average) under
N-deficient conditions. Diatoms also accumulate lipid
as a result of Si limitation, and the average lipid content
for diatoms limited for Si relative to N deficient conditions was 41%, which was 28% higher compared with
all species and 13% higher when compared within the
diatoms [99] .
In terms of neutral lipid (TAG), Si-deficient cultures of C. cryptica were reported to contain 64% on
an ash-free dry weight (AFDW) basis, compared with
32% in Si-replete cultures, and Cylindrotheca fusiformis
contained 57% in Si-deficient cultures and 17–20% in
Si-replete cultures [201] . A wide variety of TAG composition has been identified in diatoms, with fatty
acid (FA) side-chains ranging from 14:0/16:0/16:1 to
16:1/20:5/22:6; however, there were ten predominant
species, which were enriched in C14 and C16 [100] .
Diatom FAs are among the most highly enriched
in C14 chain lengths compared with other classes of
algae [98] , especially chlorophytes, which appear to lack
this particular length [101–103] . Reports range from 4 to
32% C14:0 of the total FA composition for diatoms
[98,103,104] . C16 FAs are highly represented in diatoms
and C16:0 and C16:1 combined can represent 65–70%
of the total FA, with C16:1 being predominant. C18
lengths are poorly represented in diatoms, but can be
a predominant form in chlorophytes, especially polyunsaturated forms [98,102] . In general, shorter chain
lengths are desirable to improve cold-flow properties
of biofuels [105] and saturated FAs are more desirable
future science group
because they increase the ignition quality (cetane
number) of the fuel [106] .
A critical point to make regarding measurement of
total lipid or TAG in diatoms is that DCW measurements will underestimate the actual amount of lipid
in terms of percent cellular carbon when compared
with a similar measurement in other classes of algae
because of the contribution of the noncarbonaceous
silica cell wall. Comparison of eight different diatom
species indicated ash contents from 49 to 59% [107] ;
further, diatoms had an average of twice the ash content of a variety of nonsilicified algae and four times the
content of two Chlamydomonas species examined [97] .
The silica content of three diatom species was measured
at 22–47% of the dry weight [108] . Factoring this into
DCW measurements, this suggests that lipid content
in diatoms on a per cell carbon basis is substantially
higher than usually reported. Illustrating this point is a
comparison of Si-induced lipid accumulation in C. cryptica, in which total lipid based on DCW was 42% [97]
compared with 54.1% when calculated on an AFDW
basis [109] . The data suggest that diatoms are at least
on par with, if not superior to, other classes of algae in
terms of their ability to accumulate lipids.
ƒƒ Other valuable hydrocarbons, EPA, DHA &
isoprenoids
Diatoms are unusually highly enriched for eicosapentaenoic acid (EPA; C20:5, w-3), which is not prevalent
in chlorophytes [98,103,104] . Fish do not naturally produce
EPA, but obtain it from algae they ingest, to which diatoms should contribute significantly. Docosahexaenoic
acid (DHA) levels in diatoms are less than EPA, on the
order of 5% of total lipid [98,103,104] . Diatoms contain
a rich variety of carotenoids in the form of b-carotene,
as well as a number of oxygenated derivatives called
xanthophylls. These include fucoxanthin, neofucoxanthin, diadinoxanthin and diatoxanthin, some of
which play important roles as accessory pigments and
photoprotective agents in the light-harvesting complex
of the photosystems [110] . Reports of diatom carotenoid
content ranges between 0.07 and 0.6% of the dry
weight (0.7–6.3 mg pigment/g dry wt), with pennates
exhibiting a higher content than the centrics that were
examined [108] . Some diatom xanthophylls are used
commercially for their color and antioxidant properties [111] , and it should be possible through recombinant techniques to dramatically increase production of
specific desired xanthophylls in microalgae that naturally produce these pigments. Diatoms possess both
the 2-C-methyl-d-erythritol 4-phosphate (MEP) or
nonmevalonate pathway for carotenoid synthesis, and
the mevalonate pathway (MEV) for synthesis of sterols,
sesquiterpenes and triterpenoids [112,113] .
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229
Review Hildebrand, Davis, Smith, Traller & Abbriano
Key term
Si requirement: The need by diatoms
for an additional nutrient (silicon for cell
wall synthesis) relative to other classes
of algae. Silicon limitation will induce
lipid accumulation in most diatoms.
ƒƒ Antipathogenic or medical
applications of extracted diatom
components
Although not a major field of
research, added-value chemical
components of diatoms have been
described. These include antibacterial and antifungal compounds [114–116] , which are present in FA extracts [117,118] . Antibacterial activity extracted
from Skeletonema costatum was shown to inhibit the
growth of Vibrio, a pathogen of fish and shellfish [118] .
Other extracts of S. costatum and Haslea ostrearia demonstrated anti-tumoral activities against human lung
cancer and also had anti-HIV effects [119–121] . Highly
branched C25 isoprenoidpolyenes (polyunsaturated sesterpenes oils orhaslenes) were identified as being responsible for these activities [122] . A recent report documented
that marine benthic diatoms contain compounds able to
induce leukemia cell death and modulate blood platelet
activity [123] suggesting that diatoms may represent a
richer source of interesting bioactive compounds than
previously recognized.
ƒƒ The advantages of Si limitation-based lipid
accumulation induction
As far as has been documented, diatoms accumulate
TAG only under limitation conditions. Published literature has shown that TAG accumulates under either
Si or N limitation [98,109] , and in Figure 4 we present a
comparison of these conditions with a treatment where
the cells were exposed to the microtubule-based cell cycle
inhibitor nocodazole. There are several important points
to consider from this. First is that only cell cycle arrest,
Change relative to day 0
50
Si
40
N
Nocodazole
30
20
10
0
0
1
2
Days
3
4
Figure 4. Neutral lipid accumulation using BODIPY
fluorescence in Thalassiosira pseudonana comparing
treatment with nocodazole, or limitation for Si or N.
Cultures were harvested from exponential phase
and inoculated into fresh medium for the various
treatments. The nocodazole treatment was done in a
separate experiment from Si and N.
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Biofuels (2012) 3(2)
and not nutrient limitation, is required for induction of
TAG accumulation in diatoms (Figure 4) . This has been
substantiated by other experiments in our laboratory
in which cell cycle stage is directly measured, and the
results demonstrate that TAG does not begin to accumulate until arrest has occurred [Smith SR et al. Unpublished
Data] . Often, it is stated in the literature that TAG accumulation occurs only under nutrient stress conditions;
however, experiments in our laboratory indicate that,
for diatoms stopping the cell cycle, even under nutrientreplete conditions is sufficient (Figure 4) . A second point is
that TAG accumulation under Si limitation lags slightly
behind nocodazole treatment, but reaches and eventually
exceeds the maximum level (Figure 4) . The difference in
induction start points may be related to a longer time
required to halt the cell cycle under Si limitation. The
third point is that TAG accumulation under N limitation lags substantially in timing and level of induction
compared with nocodazole and Si. After a very long limitation period, abundant lipid droplets will form under N
limitation, but only several days after similar sized droplets are seen under Si limitation [Traller JC, Unpublished
Data] . The reason for the differential response comparing
Si with N limitation is likely due to the differences in
extent of intracellular storage of the two elements. N is
stored intracellularly in a variety of forms and can be
accessed over time, resulting in continued growth even
after cells are placed in a N-free medium. Si is not appreciably stored in diatom cells, and hence placement of cells
into a Si-free medium results in relatively rapid arrest
and, therefore, a faster onset of TAG accumulation.
In terms of a production scenario, a lag time for
induction of TAG accumulation would be unfavorable – increased time means decreased overall productivity. Thus, N limitation would not be desirable for
diatoms, especially when Si limitation is an option.
For this reason, it is a valid question whether diatoms
without a Si requirement, such as P. tricornutum, would
be competitive in a production system. The rate of
TAG accumulation in chlorophytes resulting from
N limitation varies among species. In C. reinhardtii,
experiments on wild-type, starchless and complemented
mutants showed that TAG levels increased on the order
of twofold within 24 hours [124,125] , suggesting a more
rapid response under N limitation than with diatoms.
Consistent with this, growth arrest in C. reinhardtii
under N limitation appears to be fairly immediate [126] .
In C. vulgaris, growth arrest was not immediate, and
increased cell number was evident 2–4 days after placement in a N-free medium [126] . To our knowledge, no
correlation between TAG accumulation and cell cycle
arrest has been reported in chlorophytes, although correlation has been made between life cycle stage and TAG
accumulation [101] .
future science group
The place of diatoms in the biofuels industry Review
ƒƒ The detrimental effects of N limitation
In addition to the time consideration, there are other
drawbacks of having to rely on N limitation to induce
TAG accumulation. There are numerous detrimental effects of N limitation on photosynthesis [127,128] ,
which include decreased chlorophyll, alteration of thylakoid stacking, reduction in PSII cross-section and
decreased energy transfer from the LHC to reaction
centers. N limitation reduced photosynthetic rates
and acetate uptake in Chlamydomonas [125] and LHC
protein content decreased 10–40-fold, contributing
to chlorosis [129] . Chlorophyll levels typically decrease
under N limitation, which does not occur under Si
limitation in diatoms [127,130] . N limitation also dramatically reduces ribosome content in C. reinhardtii
[131] . N-deprived microalgae typically have reduced
protein content; for P. tricornutum, N starvation led
to a decrease in abundance of RuBisCO, especially the
small subunit [132] . De novo protein synthesis is necessary for an increase in acetyl CoA carboxylase activity,
the first committed step in FA biosynthesis [100,133] .
General protease activity was increased in a diatom
and Dunaliella tertiolecta under N limitation [99,134] . In
terms of transgenic approaches to improve productivity, because of the overall decrease in protein synthesis
under N limitation [127] , any expressed proteins are
likely to be less effective than desired. The combined
effects suggest that N limitation is truly stressful to
the cells, which raises the question of how cell ‘health’
is related to its ability to synthesize and accumulate
TAG. In diatoms, Si limitation has little direct effect
on other aspects of cellular metabolism than the cell
cycle [135] ; for example, protein and RNA synthesis are
not appreciably affected [136] .
ƒƒ The ease of extraction of lipid from diatoms
On occasion we have heard comments that it must be
more difficult to extract lipid from diatoms because of
their solid cell walls. This is not true. Although cell
wall components are solid, the overall structure is composed of discrete siliceous substructures that are joined
together by organic molecules. The individual parts are
relatively easily disrupted, and organic solvents readily
extract lipid and pigments [137,138] . The diatom cell wall
is actually easier to break open than the wall of some
green algae; for example, RNA isolation procedures
for diatoms involve simply vortexing cells in the presence of Tri Reagent [139] , whereas for some green algae
frozen cells have to be ground in a mortar and pestle
prior to extraction. Furthermore, pigments are easily
extracted from diatoms and blue-green algae using 90%
acetone, whereas harsher solvents are required for the
extraction of pigments from coccoid green algae [140] .
In a biofuels production setting, where the energetic
future science group
cost of extracting lipid is high, diatoms and other
more easily extractible algae may provide a significant
economic benefit.
ƒƒ Settling rates, flocculation, harvesting &
shear stress
One of the most expensive aspects of microalgal bio­fuels
production is harvesting and dewatering; the need to
concentrate a small percentage of solids from a much
larger volume of water [141] . Factors that would reduce
these costs include increasing settling rates or the ability
of microalgal cells to flocculate. As stated previously,
diatoms are major exporters of organic carbon from
the ocean surface due to their increased settling rates
relative to other phytoplankton [142] . The efficiency of
this settling rate is likely to be at least partly dependent
on the shape of the diatom. One report documented
that elongated pennate diatoms were problematic in a
water treatment facility because they did not settle well
in the facility’s sedimentation tanks, causing clogs in
the downstream sand filters [143] . However, diatom cell
walls are covered with organic material having properties similar to other algal cell walls, and respond well
to both cationic and anionic chemical flocculation
treatments [144] . Further, diatoms are known to flocculate naturally and chain-forming marine diatoms can
aggregate into centimeter-sized flocs in as little as 24 h
[145] . The ability of diatoms to naturally flocculate is
species-specific [146] , facilitated by a variety of mechanisms ranging from direct adhesion to the cell surface
production of exopolymers [147] . Some species may even
produce anti-flocculation compounds [147] . Increased
exopolymer secretion in conjunction with cellular protuberances that trap other particles is a common sinking
mechanism in marine species, which is often triggered
near the end of a bloom and generates sinking rates
of 100 m day-1 or more [148] . These data indicate that
understanding diatom self-flocculation strategies may
be of value in the context of biofuels production.
Diatoms are well adapted to turbulence, and in fact
are more productive in high turbulence conditions than
other classes of marine microalgae [149] . However, there is
some apparent contradiction in the literature concerning
this point, in that some studies indicate that diatoms are
especially sensitive to shear during cultivation [150,151] .
In one report, the authors concluded that micro-eddies
on the scale of the cell size were responsible for shear
stress [150] . A suggestion was made that diatoms may be
especially sensitive to shear stress because of their rigid
silica walls [152] ; however, the particular species used in
this study was predominantly unsilicified. Another study
examined the passage of two diatom species through
pumps and valves and concluded that the extent of cellular damage was related to operational parameters, such as
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231
Review Hildebrand, Davis, Smith, Traller & Abbriano
Key term
the pressure drop and the rotational
speed of the pump [153] . Cultivation
in our laboratory has revealed that
some species are impervious to
highly turbulent mixing, whereas
others are sensitive to modest agitation, suggesting that species-specific
differences need to be taken into account with regard to
shear stress; such differences have also been reported in
the literature [150,154] . The data suggests that the issue is
not with diatoms as a class, but particular diatom species
subjected to particular turbulence conditions.
Environmental flexibility: The ability of
a microalgal species to adapt and grow
in different environmental conditions,
which is relevant to the development of
biofuels production systems.
ƒƒ Susceptibility to predators or pathogens &
resistance to contamination
In spite of the protective cell wall, diatoms are not
immune to predators and pathogens. A major class of
diatom predator in the ocean is copepods, which are
small crustaceans [155] . Interestingly, diatoms have apparently developed both physical and chemical defenses
against copepods. A study demonstrated that diatom
cell wall architectures were optimized to resist crushing [156] , in this context by copepod jaws, and diatoms
also produce aldehyde derivatives that have been implicated in reducing copepod reproductive viability [155] .
Apofucoxanthinoid compounds (derived from fucoxanthin) produced by diatoms act as copepod feeding deterrents at concentrations of 2.2–20.2 ppm [157] . Small flagellates also feed selectively on diatoms [158] . In general,
diatoms represent a bulky food for ingestors and their
cell walls must be compromised during ingestion or else
the intracellular contents are not completely accessible.
Chytrid fungi infect diverse prokaryotic and eukaryotic
algae, including diatoms [159] . Phytomyxea (plasmodiophorids) parasites have also been shown to parasitize diatoms [160] . Diatom viruses were first described in 2004
[161] and are either ssRNA or ssDNA viruses that infect
the genera Rhizosolenia or Chaetoceros [162] . It is likely
that the measures to deal with predation and pathogens
that are being considered for other classes of algae will
need to be applied to diatoms as well for biofuels production. In terms of cross-contamination of cultivation
systems or environmental contaminants, one advantage
of the defined shapes and sizes of diatoms is that it is
generally easy to use microscopy to identify differences
between diatom species, as well as to distinguish diatoms
from other microalgae. The generally higher nutrient
uptake rate of diatoms compared with other classes of
algae (as mentioned previously) could also contribute to
reduced contamination.
ƒƒ Genomic & transgenic aspects of diatoms
Genetic modification is likely to play an important role
in the development of algal biofuels, both as a research
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Biofuels (2012) 3(2)
tool and in possible field applications once appropriate
regulations are in place. Diatoms are well established in
terms of genomic and transgenic capabilities. There are
currently four available diatom genome sequences, for
T. pseudonana, P. tricornutum, Fragilariopsis cylindrus
and Pseudo-nitzschia multiseries. Of the four, F. cylindrus
and Pseudo-nitzschia multiseries may not have biofuels
relevance, because neither grows at a fast enough rate for
a production system. Sequenced genome sizes in diatoms
vary from compact at 27 Mbp to larger on the order of
300 Mbp, and GC content is close to 50% [8,10] . The latter point is important because many genes already developed for mammalian or yeast purposes (e.g., eGFP) can
be used directly in diatoms with no modification, such
as codon optimization [163] . Another important point is
that there is no evidence for gene silencing in diatoms, as
occurs in some chlorophytes [164] . In one study, a transgenic diatom was cultured in the absence of antibiotic
selection for 1 year and retained resistance [165] .
Currently diatom transformation is limited to
the biolistic approach [165,166] , which is effective, but
in­efficient. Higher efficiency methods would be desirable; for example, to enable saturation screening of
insertional mutants or to complement mutations by
introducing a library of genes. Integration of introduced
DNA occurs randomly in diatom genomes [162] , which
can result in different levels of expression depending on
the site of integration. This is not necessarily negative;
on the one hand, more involved screening is required
to identify useful transformants, but the variation in
expression can be useful in obtaining a graded response.
Homologous recombination has not been reported in
diatoms, so gene replacement is not yet an option.
Promoters that drive overexpression of genes have been
identified [163,165,166] , as well as a regulated promoter that
can be turned on or off by changing the N source in the
growth medium [167] . Gene knockdowns have also been
accomplished using RNAi approaches [168] . Although
there is room for improvement, a complete set of basic
genetic manipulation tools are available for diatoms.
ƒƒ Diatom metabolic diversity could provide
abundant targets for bio-engineering
The evolutionary divergence of centric and pennate
diatoms occurred at least 90 million years ago (based
on the fossil record), yet their genomes differ from one
another to the same extent as those of fish and mammals, which diverged approximately 550 million years
ago [8] . The relatively rapid rate of diatom evolution
has been at least partially attributed to the acquisition
of horizontally acquired genes, which are thought to
confer novel cellular and metabolic functions, and are
drivers of diversification [8] . However, there are also
substantial differences between diatoms in the targeting
future science group
The place of diatoms in the biofuels industry Review
Perceived disadvantages of diatoms in
large-scale cultivation
The vast majority of diatoms require Si for growth,
and thus a major difference between cultivating diatoms and other classes of algae is the requirement to
add soluble Si to the medium to support growth. The
presence of an additional nutrient can be perceived as a
drawback in a culture system because of increased cost
and, as in the case of Si, ancillary problems that could
arise because of its aqueous chemistry. Complete details
of the aqueous chemistry of Si can be found in Iler [75] .
As a brief summary, soluble Si is present in aqueous
solution as silicic acid, Si(OH)4, which is the predominant form at pH 8.0, with a small fraction (3%) of the
monoanion Si(OH)3O-. The solubility of silicic acid is
limited at neutral pH to approximately 2 mM, above
that concentration it begins to autopolymerize to first
form multimers, which eventually coalesce into solid
silica, SiO2, which can precipitate from solution [75] .
Silicic acid can form complexes with metal ions, and
magnesium silicates are especially prone to precipitation
[75] . Another perceived problem with silica relates to the
important distinction between the amorphous silica
found in diatoms and crystalline silica found in asbestos. Although chemically identical, their toxic effects
are completely different. Needle-shaped crystalline
silica is a causative agent in silicosis, in which scarring occurs in the upper lobes of the lungs. Amorphous
silica is harmless to all organisms other than insects
(see below) when breathed, ingested or contacted; thus,
large-scale diatom cultivation will not produce a hazardous silica product.
There are advantages to the Si requirement for diatoms in cultivation. As discussed previously (Figure 4) ,
Si limitation results in faster TAG accumulation than
N limitation, with attendant reduction in production
time. An interesting concept is whether a Si limitation
lipid strategy actually requires more N than a species
that accumulates under N limitation. There are two
considerations regarding this: increased neutral lipid
future science group
productivity under Si limitation (Figure 4) may outweigh the extra cost of N, and N could be included during the Si-limitation phase at levels much lower than
used for growth to prevent the detrimental effects of
N limitation as discussed previously, but not in excess
of that, thus minimizing cost. Another benefit of the
diatom’s Si requirement is that biomass accumulation
can be precisely regulated by the amount of Si added to
batch cultures. Many diatom species have well-defined
silica content in their cell walls; thus, there is a strict
dependency on how many cell divisions can occur with
a given starting amount of Si in the medium. This
is illustrated in Figure 5, where batch cultures of C.
cryptica were grown with different starting concentrations of Si. Growth arrested in each culture on day 7,
and the resulting culture density was precisely in the
ratio of the starting amount of Si in the media. Such
control could be highly valuable in a large-scale culture
system because biomass attainment would both be predictable and controllable. Similar control may not be
likely with other nutrients, such as N, given the issue
of intracellular storage and the abundant presence of N
sources in the environment (including N fixers). There
are scenarios where Si limitation may be problematic in
a production setting; for example, Si-containing dust
could blow into an open system and perturb the control. However, the effects of this type of contamination
should also be predictable once the rate of dissolution
of airborne Si into the medium is determined. It should
be noted that it is not likely that all diatom species will
exhibit such precise control of biomass relative to Si
concentration. Heavily silicified diatoms are known to
reduce in size with succeeding generations (eventually
2.5
Cells per ml (× 06)
and homology of core genes involved in highly conserved eukaryotic pathways and processes. These
include differences in the homology and distribution
in enzymes of carbohydrate metabolism, the pentose
phosphate pathway and carbonic anhydrases [59,68,169–
171] . Differences in these core pathways are likely to
affect metabolism and/or cellular energetics. These differences indicate that the trophic and environmental
flexibility found within the diatoms has a genetic basis.
Diversity in the core metabolism of diatoms means
that there are multiple possibilities for manipulation
of enzymes and pathways to improve growth and lipid
accumulation characteristics.
100 µM
200 µM
2.0
300 µM
400 µM
1.5
1.0
0.5
0
1
2
3
4
5
6
Days
7
8
9
Figure 5. Control of biomass accumulation in
cultures of Cyclotella cryptica by altering the starting
concentration of silicon. Incremental amounts of
silicic acid were added to the cultures at day 1 and
growth followed thereafter. On day 7 when all cultures
were limited, the ratio of cell numbers to starting
concentration were in exact correspondence.
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Review Hildebrand, Davis, Smith, Traller & Abbriano
triggering the sexual cycle), which leads to a mixed size
population of cells with varying amounts of silica in
their walls. An important point is that size reduction
is not universal in diatoms, and in most laboratory
species there is no evidence of size reduction, and have
documented reasons why it does not have to occur [172] .
Given its limited solubility (0.07% in water), the
method of delivery of Si to a culture system deserves
some consideration. Most production systems are
maintained at or above pH 8, which is favorable for
silicic acid solubility; however, there are still limits. A
maximum of 2 mM Si could be added to a batch culture, but in addition to pushing the solubility limit,
such high concentrations could affect the growth of
diatoms. Environmental concentrations of silicic acid
are rarely above 100 µM, and we have observed detrimental effects on some species in laboratory cultures at
400 µM. Fortunately, the amount of free silicic acid in
a culture can be readily measured, either by estimating
the amount based on biomass accumulated (Figure 5) ,
or by direct chemical measurement using a colori­metric
assay [173] . By regular monitoring, one could add Si
when needed to promote high biomass accumulation
without detrimental side effects.
A valid concern is what would be the added cost of
including Si as a component of the growth medium.
There is a lack of publically available information on
Si’s projected costs; in nine cost ana­lysis publications
over the past 2 years, only one found one that mentioned the words Si or silica, and there were no cost
figures for Si included [141] . Our discussion is therefore
limited to general concepts. First is the availability of
precursor raw material; Si is the second most abundant
element on the earth’s surface, and so supplies are not
limited. The true cost will be in the conversion of precursors to silicic acid. A variety of types of silica ranging
from crystalline quartz (e.g., sand) to amorphous diatomaceous earth will dissolve in water, with amorphous
forms dissolving faster [75] . Second is the cost related to
the recycling of silica from spent medium or biomass.
Because diatom silica readily dissolves at high pH [75] ,
recovery of silica from lipid-extracted diatoms should
be feasible, without detrimental effects on other cellular
components, such as protein. Therefore, recovered Si
could be recycled repeatedly, since the chemical form of
dissolved Si will not be altered, as might happen with
other nutrients such as N. A third factor is the possibility of using diatom silica as a value-added product,
which is discussed below. The overall cost of adding Si
to a production system will depend on these factors and
has to be balanced against potential increases in productivity using diatoms in a Si limitation approach. To
date, no studies have addressed these issues in a quantitative way, which obviously needs to be done to evaluate
234
Biofuels (2012) 3(2)
the possibilities and to provide impetus for potentially
improving processing methods to reduce costs.
Diatom silica generated in a production system can
be considered as a marketable product. Diatomaceous
earth is already widely used in commercial processes
and products. Due to the porous nature of diatom frustules, they are excellent filtering materials for very fine
particles, and are primarily used in pool filters and for
filtration of beer and wine. Diatom silica is also highly
hygroscopic, and it is used as an insecticide because its
contact with the cuticle stimulates desiccation of the
insect in conjunction with perforating the cuticle with
sharp-edged small particles. It is also used as a neutral
anthelmintic (dewormer) in mammals. The abrasive
nature of microscopic diatom particles is taken advantage of in metal polishes and toothpastes. Diatom silica,
which is essentially glass, has optical properties, and
when added to paint, makes it flat. Diatomaceous earth
is added to plastic wrap to separate the layers slightly to
prevent them sticking tightly to each other. Dynamite
is a stabilized form of nitroglycerin, which is absorbed
into diatomaceous earth.
Mined diatomaceous earth is usually a mixture of
diatom species, and so can be relatively heterogeneous.
It is possible that diatom silica isolated from a monoculture pond may have more consistent properties.
One possible future application of diatom silica is in
nanotechnology. Diatoms make nanostructured silica
in a variety of shapes that cannot be achieved by current synthetic materials approaches. Silica has limited
properties as a material but several approaches have
been developed by which diatom silica can either be
directly converted or used as a template to generate a
completely different chemistry while maintaining the
nanoscale structure [174] .
Diatoms are likely to have other added-value applications. They are currently used as small percentage
supplements to animal feed, and we are aware of studies
evaluating the use of diatoms as a larger component of
animal feed, ranging from fish to cattle. EPA from cult­
ivated diatoms is less expensive than from fish sources
[175] . Other applications, such as amino acids for cosmetics, antibiotics and antiproliferative agents, are at
early stages of development [175] .
Conclusion
A question commonly asked is, “If diatoms have such
desirable properties and potential advantages, then
why aren’t they more highly represented in biofuels
research and production projects?” After considering
their attributes as described in this article, it is puzzling.
Two possible explanations come to mind. One is the
long history of the study of chlorophytes due to their
intimate relationship with terrestrial plants (especially
future science group
The place of diatoms in the biofuels industry Review
related to photosynthesis) and our food supply. Another
is related to the fact that diatoms dominate marine environments, whereas chlorophytes and plants dominate
terrestrial environments. The ocean is a difficult system
to study, and there are fewer research institutions with
a marine focus than those dedicated to terrestrial environments; this imbalance may be an additional reason
why the virtues of diatoms have been under appreciated.
Generalized statements about the properties of ‘algae’
are also often heard. It is hoped that this review has
highlighted the substantial differences not only between
different classes of microalgae (specifically between diatoms and chloro­phytes), but between different species
within a class. Generalizations are not only inaccurate,
but obscure the rich diversity of microalgae, which is
a ‘value-added product’ in itself. At this early stage in
the development of algal biofuels, little is known about
the fundamental mechanisms involved in carbon fixation, flux and partitioning in microalgal cells, nor are
scientists well versed in how to grow microalgae at scale.
The deployment of algal biofuels on a production scale
will involve largely uncontrollable and only partially
predictable environmental conditions; thus, development of strains with diverse environmentally related
productivity attributes will be necessary. For these reasons, it is valuable to invest­igate diverse classes of algae as
research models and as potential production organisms.
In a practical sense, this will define what is possible in
terms of environmental adaptability and productivity,
and allow a survey of the gene pool, which could be
used to improve production species. As a class of algae,
diatoms have all of the requisite attributes to be included
as important contributors to the development of algal
biofuels technology.
Future perspective
There has been substantial progress in the development
of algal biofuels in the past few years. This may not yet
be reflected in the publication record, because of the lag
time in getting data published and corporate policies
to protect data; however, at algal biofuels meetings it is
clear that there is an undercurrent of excitement about
things moving forward. The two major challenges in
the develop­ment of this technology are the development
of the “biology” to understand and improve factors that
result in consistently high productivity of microalgal
strains and development of large-scale culture systems
that can grow algae and produce fuel precursors economically and at predictable production levels. Of the two
challenges, understanding the biology is likely to be more
straightforward, given the powerful tools already available
to investigate and manipulate cellular metabolism. After
an initial phase, over the next several years, of understanding and manipulating metabolic factors involved in fuel
Executive summary
Evolutionary aspects of diatoms
ƒƒ Diatoms have a different evolutionary history than chlorophytes, which resulted in fundamental differences in cellular organization and
metabolic processes.
ƒƒ On geologic timescales diatoms have become the predominant carbon fixers in the ocean.
Diatom productivity
ƒƒ Diatoms out-compete other classes of algae for growth, which may result from superior nutrient acquisition and storage and
photosynthetic efficiency.
ƒƒ Diatoms are environmentally adaptable and trophically flexible.
What cellular factors contribute to diatom productivity?
ƒƒ Diatoms exhibit more efficient photosystem electron flux and increased selectivity for CO2 compared with other classes of algae.
ƒƒ Diatoms have substantial differences in intracellular compartmentation, the presence and localization of metabolic pathways and forms of
carbohydrate stored compared with chlorophytes.
ƒƒ The rate of lipid accumulation is substantially improved under silicon limitation relative to nitrogen limitation, and numerous detrimental
effects of nitrogen limitation are avoided.
ƒƒ The silica cell wall may reduce carbon requirements and energy for cell wall synthesis.
Attributes of diatoms for biofuels production
ƒƒ Diatoms have been cultivated for decades in outdoor open systems for aquaculture.
ƒƒ Diatoms naturally grow under bloom conditions – maximum growth to high density.
ƒƒ Diatoms accumulate abundant lipids, and lipid content may be underestimated unless based on an ash-free dry weight basis.
ƒƒ Diatoms produce added-value products.
ƒƒ The diatom silica cell wall may contribute to improved production through precise control of biomass accumulation and increased
settling rates.
Perceived disadvantages of diatoms in large-scale cultivation
ƒƒ Diatoms require silicon to grow, but the costs of supply, recycling and value-added products relative to productivity have not been
determined.
ƒƒ Abundant silicon supply exists, and recycling should be feasible.
future science group
www.future-science.com
235
Review Hildebrand, Davis, Smith, Traller & Abbriano
precursor synthesis in model species and initial production strains, a transition into applying these tools to a
wider variety of production strains, which will be adapted
to specific environmental conditions, is anticipated. This
may also include multispecies cultivation, in which one
or the other species is the primary producer under given
conditions. This approach will require greater understanding of the ecological aspects of microalgal growth.
Throughout the development of the organismal basis
for biofuels production, an intimate interaction with the
engineering and production aspects of large-scale cultivation will be needed.
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Diatom biofuels research in the Hildebrand laboratory is supported
by AFOSR grant FA9550-08-1-0178, DOE grant DE-EE0001222
and NSF grant CBET-0903712. SRS was supported by the
Department of Defense through the National Defense Science and
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