environmental manipulation of select algae strains for maximal oil production
Post on 08-Dec-2016
214 Views
Preview:
TRANSCRIPT
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofitpublishers, academic institutions, research libraries, and research funders in the common goal of maximizing access tocritical research.
Environmental manipulation of select algae strains for maximaloil productionAuthor(s): Travis A. Lyon , Joel G. Primm , Michael S. Wojdan , Kristopher D.Morehouse , Melanie L. Grogger , Simina Vintila , and Donald V. VeverkaSource: BIOS, 84(1):21-29. 2013.Published By: Beta Beta Beta Biological SocietyDOI: http://dx.doi.org/10.1893/0005-3155-84.1.21URL: http://www.bioone.org/doi/full/10.1893/0005-3155-84.1.21
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in thebiological, ecological, and environmental sciences. BioOne provides a sustainable onlineplatform for over 170 journals and books published by nonprofit societies, associations,museums, institutions, and presses.
Your use of this PDF, the BioOne Web site, and all posted and associated contentindicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.
Usage of BioOne content is strictly limited to personal, educational, and non-commercialuse. Commercial inquiries or rights and permissions requests should be directed to theindividual publisher as copyright holder.
Research Article
Environmental manipulation of select algae strainsfor maximal oil production
Travis A. Lyon, Joel G. Primm, Michael S. Wojdan, Kristopher D. Morehouse,
Melanie L. Grogger, Simina Vintila, and Donald V. Veverka
Department of Biology, United States Air Force Academy, Colorado Springs, CO 80840
Abstract. Consideration for the use of microalgae as a feedstock for commercial biofuels
production began in the 1970s. While high production costs and inefficiencies impeded private
industry scale up to meet national energy needs, recent energy supply concerns and market
instability has revitalized this alternative energy process. A key factor in using microalgae comes
from efficiently harvesting algae oils which are chemically similar to fossil fuel oils. Technological
advances have been made to genetically engineer microalgae to accumulate oils but these
procedures are time consuming and expensive. Others have made progress in using natural
processes in the environment to cause algae to produce oils. Considerable research has shown
determining the appropriate mix of light, temperature and CO2 can foster oil accumulation in select
microalgae strains. Using an experimental design, the freshwater algae, Chlorella pyrenoidosa and
Ettlia sp. were subjected to various environmental conditions to investigate effects on oil
production. When exposed to a higher irradiance level (400 lE), C. pyrenoidosa achieved a
significantly higher cell count leading to an increase in lipid productivity. Under media
manipulation effects, the cell count for nitrogen-limited cultures was significantly lower than that of
cultures grown on replete medium. Nitrogen limitation seems to negatively impact lipid
productivity under our experimental conditions. Supplementation with 2% CO2, led to an increase
in growth after seven days of incubation; however, growth decreased subsequently to a slower rate
as compared to growth with air sparging after 12 days of incubation. There appeared to be a degree
of variability with regards to lipid ratios, with increases in neutral lipids in cultures exposed to
higher levels of irradiance, nitrogen limitation and additional CO2. Although analysis showed that
environmental manipulation is feasible in terms of oil accumulation for these select strains, further
study is warranted in investigating other oil-accumulating microalgae strains for commercial
production.
Introduction
Investigation involving the use of micro-
algae species for the purposes of produc-
ing biofuels began in earnest at the onset
of this country’s energy crisis in the 1970s (Hill
et al., 2006). The term microalga describes a
very diverse group of thousands of organisms
with different types of cells displaying a wide
spectrum in their potential to produce biofuels
or precursor molecules to biofuels. For example,
certain algae accumulate lipids which can be
chemically modified into hydrocarbon fuels or
produce hydrogen, which can be subsequently
used for power generation in vehicles, planes
and other fuel-consuming devices/equipment.Correspondence to: simina.vintila@gmail.com
21BIOS 84(1) 21–29, 2013
Copyright Beta Beta Beta Biological Society
These photosynthetic microorganisms have the
potential to compete favorably with other oil
crops/feedstocks such as soybeans and oil palm.
A major advantage microalgae have over other
types of oil crops is their potential to produce
higher yields of oil per hectare of land. Many
have reported that microalgae have the capabil-
ity to produce a several-fold increase in oil per
acre over that seen in soybean production
(Chisti, 2008; Hu et al., 2008; Sheehan et al.,
1998). Some estimate that even with palm oil,
considered one of the most productive of the oil
crops, production would fall significantly short
of current U.S. biodiesel demands (Chisti,
2007).
The problem largely resides with available
land to grow the requisite amounts of crop oil
needed for conversion to biofuels. Considerable
agricultural land (used for food production)
would be needed to support oil crop require-
ments, severely impacting U.S. capability in
terms of providing cereal grains and livestock.
In contrast, depending on the cultivation
method, microalgae could be grown on margin-
al lands (non-arable, arid, etc.) not suitable for
conventional agricultural practices (Chisti,
2007). Further, many species of algae flourish
in saline/brackish water or coastal seawaters and
can utilize growth nutrients from wastewater
such as nitrogen and phosphorus. Even when
grown under environmentally controlled condi-
tions (photo-bioreactors), microalgae outper-
form land- raised crops by several fold.
Although the literature (NREL, 2006; Sheehan
et al., 1998; Shelef et al., 1984) provides a
myriad of questions that should be addressed to
feasibly use microalgae for renewable and
dependable energy sources, more recent inves-
tigation (Durrett et al., 2008; Hu et al., 2008)
has focused on adequately building sufficient
algal stocks through strain selection (identifica-
tion/screening), biomass accumulation (ponds
vs. photo-bioreactors), production methods
(lipid yield) and harvesting (extraction meth-
ods).
While tens of thousands of species of algae
have been identified, those belonging within the
green algae taxa (Chlorophyceae) appear to hold
the greatest promise (Hu et al., 2008). Green
algae have a tendency to produce more starch
than lipid but have high growth rates at higher
temperatures and lighting conditions. Keeping
in mind the goal to cultivate high quantities of
select microalgae feedstocks to produce oils,
there are some green algae (Botryococcus
braunii and Nannochloropsis oculata) that have
been cited in the literature as having higher than
usual lipid yields (B. braunii reported yields of
up to 80% dry cell weight and Nannochloropsis
species have provided yields nearing 70%)
(Chisti, 2007; Hu et al., 2008). Lipids produced
by these strains contain very long carbon chains
(C23-40), similar to those in petroleum, and
make attractive feedstocks for investigation and
development (Banerjee et al., 2002, Metzger
and Largeau, 2004). Diatoms (Bacillariophyta)
have also been investigated as potential feed-
stocks; however, their nutrient needs are much
different from other algae in that silicate is an
important nutrient for growth.
Determining optimal strains requires in-
depth understanding of the microalgae lipid
production process. Given the pressing eco-
nomic realities associated with biofuels produc-
tion, work that uncovers key environmental
factors which promote cellular lipid develop-
ment would be of great value. While the
literature varies as to what causes the cells of
algae to produce high quantities of cellular
lipids, most agree that stress (nutrient depriva-
tion, varying temperatures, or high light condi-
tions) predisposes the microalgal cell to move
toward the triacylglycerol (TAG) pathway (Hu
et al., 2008). Li et al. (2011) found that
Scenedesmus sp. could grow under a wide
range in temperatures but reported 208 C as the
optimal temperature for greatest level of bio-
mass and lipid production. Additional work by
Lin and Lin (2010) revealed that certain
nitrogen sources can alter fatty acid profiles in
another freshwater algae strain similar to
Scenedesmus rubesens. Weldy and Huesemann
(2007) investigated the results of nitrogen
limitation and light intensity on Dunaliella
salina cultures cultivated within a closed photo-
bioreactor system. Manipulating light and
nutrient levels between comparable systems,
authors found that while nutrient deprivation
22 BIOS
Volume 84, Number 1, 2013
and increased light intensity can increase lipid
content as a percentage of dry weight, total lipid
productivity is much greater under conditions
where Dunaliella salina is nutrient-replete
under high light intensity. While percent lipid
content is an important factor, Weldy and
Huesemann (2007) argue that greater amounts
of oils can be accumulated by healthier
Dunaliella salina cultures via increased cell
numbers (maximizing biomass). Total lipid
productivity should thus be of great importance
to private industry when depending on econo-
mies of scale to ensure profitability and
efficiency of operations.
A number of studies have shown that in
many microalgae cellular lipid accumulation,
generally in the form of triacylglycerides
(neutral lipids), is positively affected by stress
conditions such as nutrient deficiency or high
irradiance (Hu et al., 2008; Roessler, 1990). It
has also been shown that changes in growth
conditions (light intensity, temperature) and/or
nutrient composition can not only affect growth
and lipid quantity but also lipid composition
(Greenwell et al., 2010; Hu et al., 2008; Lin and
Lin, 2010). It should be noted that stressful
growth conditions may actually decrease total
lipid productivity through a negative effect on
growth. However, according to Rodolfi (2009),
few studies have investigated the effect of
different environmental parameters on lipid
productivity (Rodolfi et al., 2009). Nutrient
composition and ratios determine where the
cellular metabolism is directed. Key nutrients
that reportedly affect lipid yield include carbon
(in the form of carbon dioxide), nitrogen and
phosphorus. The carbon/nitrogen (C/N) ratio of
the cell generally determines if energy is to be
spent or stored (in the form of starch or
triacylglycerols). Changes in irradiance levels
primarily affect cells by increasing or decreas-
ing the rate of photosynthesis and subsequently
the rate of carbon fixation, although it is
important to keep in mind that cellular respons-
es to irradiance changes are largely species-
dependent (Guschina and Harwood, 2006).
The manner in which algae partition their
carbon sources towards various metabolic
processes, especially for lipid accumulation,
remains unclear. Developing a balanced set of
environmental variables that promote lipid
productivity, at least for a few specific strains,
would provide insight for an additional com-
mercial process in which to cost- effectively
produce algae-produced biofuels.
This study examines the effect of irradiance,
nitrogen limitation and carbon dioxide sparging
on growth and lipid productivity of the
chlorophyte algae Chlorella pyrenoidosa and
Ettlia sp. We also examined the effect of
osmotic stress on these strains as another
potential environmental factor to ascertain algal
strain robustness under varying conditions. The
genus Chlorella is generally a freshwater alga
but, interestingly, C. pyrenoidosa has been
reported to grow in saline medium (Poyton,
1970). Using marine medium also demonstrates
the flexibility of microalgae as a biofuels
feedstock to avoid competing for freshwater
resources. While the effects of irradiance,
nitrogen starvation and carbon dioxide (CO2)
supplementation on total lipid accumulation
have been investigated for a number of green
algae, no study has investigated the effects of
these parameters on lipid productivity during
osmotic stress.
Materials and Methods
Growth conditions
Chlorella pyrenoidosa (obtained from Air
Force Research Laboratories, Tyndall AFB, FL)
was grown in modified f/2 medium (Guillard
and Ryther, 1962) excluding vitamins. Cultures
of Ettlia sp. (obtained from Dr. Juergen Polle,
Brooklyn College) were grown in modified
Bold’s Basal medium with 1/3 of nitrogen and
without vitamins (Culture Collection of Algae
and Protozoa, http://www.ccap.ac.uk/media/
documents/3N_BBM_V_000.pdf). For the ni-
trogen limitation experiment, Chlorella cultures
were grown at 258C and 200 lE irradiance,
either in nitrogen-replete or nitrogen-limited
medium using batch cultures, and samples were
collected 6 and 12 days after the start of the
experiment. Nitrogen limitation was achieved
by decreasing the nitrate levels 20-fold com-
pared to the original concentration in the f/2
Manipulation of algae for maximal oil production 23
Volume 84, Number 1, 2013
medium (from 8.8 to 0.44 mM). For the
irradiance experiment, batch cultures were
grown at 200 lE and 400 lE in f/2 medium
on an orbital shaker. To investigate the effects of
CO2 on growth and lipid accumulation, semi-
continuous cultures of C. pyrenoidosa and Ettlia
sp. were grown in a 14 L BioFlo 115 photo-
bioreactor (New Brunswick Scientific, Edison,
NJ) in f/2 or BBM medium, respectively, with a
media flow rate of 1 L/day (0.6944 ml/min) for
C. pyrenoidosa and 6 L/day (4.2 ml/min) for
Ettlia sp. The cultures were sparged with 2%CO2 and 98% air at 0.5 L/min.
Growth was monitored during all experiments
by cell counts using either a hemocytometer or
flow cytometer (Accuri C6 Flow Cytometer, Ann
Arbor, MI). All experiments were run in
biological triplicates (except experiment on
Ettlia sp.). Samples were taken in duplicates.
Sampling and lipid extraction
For lipid extraction, 500 ml of cell culture
was centrifuged at 6000 x g for 10 min. Cell
pellets were freeze dried and stored at -208C
until subsequent lipid extraction. Lipid extrac-
tion was performed as previously reported with
modifications (Bigogno et al., 2002). Briefly,
freeze-dried cells were suspended in 4-5 ml of a
chloroform : methanol mixture (2:1, v/v) with
0.2 g quartz particles (size 1.2-20 lm, Sigma).
Each mixture was homogenized with a needle
sonicator at 18 W for 3 minutes. For maximum
extraction, the suspension was allowed to stand
for at least 6-8 h. Each vial was then centrifuged
at 7000 x g for 10 min and the organic phase
was transferred to a new vial. Chloroform:water
(1:1, v/v) was added to rinse non-lipid mole-
cules from the organic phase and vials were
centrifuged at 7000 x g for 10 min. The organic
phase was transferred to pre-weighed glass vials
and the solvent was evaporated under N2
atmosphere. The remaining algal oil was
weighed and normalized.
Lipid analysis
Lipid classes were analyzed using automated
thin layer chromatography coupled to a flame
ionization detector (TLC-FID) system (Iatro-
scan MK6, Iatron, Japan). The lipid samples
were dissolved in 200 ll of a chloroform:me-
thanol mixture (2:1, v/v), then 1 ll of each
sample was spotted onto silicon rods. The rods
were developed using a two-bath system. The
first bath contained a mixture of hexane:diethyl
ether:formic acid (98:2:0.1, v/v/v) and rods
were developed for 20 min. The second bath
contained a mixture of hexane:diethyl ether:
formic acid (80:20:0.1, v/v/v) and the rods were
developed for 10 min. The rods were then dried
at 1208C for 2 minutes. The samples were
placed in the Iatroscan, which was set at 2 L/
min air and, 160 ml/min H2, and set to scan for
30 seconds. Samples were run in technical
duplicates. Glycerol Trioleate, Palmitic Acid,
Cholesterol, Nonadecane and Palmityl Palmitate
(Sigma Aldrich, St Louis, MO) were used as
external standards.
Statistical analysis
ANOVA followed by Tukey HSD was
carried out using the R statistical software
program, version 2.3.1 for Windows XP
(http://www.r-project.org/).
Results
The effects of irradiance, nitrogen limitation
and CO2 supplementation on growth, lipid
composition and productivity of the green alga
Chlorella pyrenoidosa were examined. In addi-
tion, the effect of CO2 supplementation was
investigated for one other green alga, Ettlia sp.
The results indicate that all three of the above
mentioned parameters affect growth and lipid
productivity.
Effect of irradiance on growth and lipid
productivity
C. pyrenoidosa cultures grown under a
higher irradiance (400 lE) as compared to
control cultures (200 lE) achieved a signifi-
cantly higher (p < 0.001) cell count (Figure 1a)
during the incubation period. At the same time,
the proportion of neutral lipids was 25 % lower
24 BIOS
Volume 84, Number 1, 2013
(p = 0.02) at 400 lE than 200 lE (Figure 1b).
While the lipid amount per cell may be lower
after nine days of incubation than for cultures
grown under 200 lE, the higher cell count
under 400 lE actually led to higher lipid
accumulation per liter of culture. Taken togeth-
er, the results indicate that higher irradiance has
a positive effect on lipid productivity.
Effect of nitrogen limitation on growth and
lipid productivity
To achieve nitrogen-limiting conditions, the
nitrate amount in the medium was reduced from
8.8 mM to 0.44 mM (a 20-fold reduction). C.
pyrenoidosa cultures were incubated for a total
of 12 days. As shown in Figure 2a, no
difference in cell density could be seen between
nitrogen-limited cultures and control cultures
for the first 6 days of the experiment (p = 0.99).
After 12 days of incubation, the cell count for
nitrogen-limited cultures was significantly lower
(p < 0.001) than that of cultures grown on
replete medium (Figure 2a). Interestingly, the
proportion of neutral lipids decreased signifi-
cantly (p = 0.004) between day 6 and day 12 of
incubation (Figure 2 b) for the control culture
and while a slight decrease could also be
observed for the nitrogen-limited culture, the
decrease in neutral lipids was not statistically
significant (p = 0.144).The proportion of
neutral lipids was also somewhat higher under
nitrogen-limiting conditions (Figure 2c). While
the relative amount of lipid did not differ
between cultures grown on replete medium
and nitrogen-limited cultures (day 6 p = 0.2;
day 12 p = 0.4), the cell counts were
significantly different (p < 0.001). When
growth rate is taken into account, nitrogen
limitation, at the level tested, has a negative
effect on total lipid productivity (lipid accumu-
lation per liter of culture).
Effect of CO2 supplementation on growth
and lipid productivity
Cultures subjected to CO2 supplementation
were grown under semi-continuous conditions.
C. pyrenoidosa cultures supplemented with 2%
CO2 displayed an increase in growth after seven
Figure 1. Cell density and neutral:polar lipid ratio at varying irradiance a) Cell densities of C. pyrenoidosa culturesgrown at 200 lE and 400 lE. b) Relative proportions of neutral and polar lipids in C. pyrenoidosa cultures grown at 200 lEand 400 lE.
Manipulation of algae for maximal oil production 25
Volume 84, Number 1, 2013
days of incubation but growth decreased and
cell densities were lower than at the start of the
experiment after 12 days of incubation (p <0.001; Figure 3a). The proportion of neutral
lipids was negatively correlated with growth
(Figure 3b). During the increase in cell growth,
the proportion of neutral lipids decreases but
increases again after 12 days of incubation
(Figure 3 b) There was however some variabil-
ity between samples and therefore the observed
changes in neutral lipid proportion are not
statistically significant (p = 0.209).
The same experiment was performed on
another freshwater alga, Ettlia sp. As shown in
Figure 4a, the response to CO2 supplementation
of Ettlia sp. cultures was similar to that of C.
pyrenoidosa. During the first 6 days of incuba-
tion, the cultures displayed an increase in
growth accompanied by a decrease in the
proportion of neutral lipids (Figure 4b). After
8 days of incubation, the growth was markedly
reduced and the cells had an increased propor-
tion of neutral lipids accumulated. While total
lipid accumulation per cell was increased at the
end of each experiment for both species, the
overall growth decreased. Considering both
total lipid accumulation per cell and growth,
the highest lipid productivity can be found 6 to
7 days after CO2 supplementation, coinciding
with the increase in growth for both species.
Discussion
The main goal of this study was to
investigate the effect of three environmental
conditions on lipid productivity for species not
previously investigated. The results are in
agreement with previous studies showing that
stress conditions seem to positively affect the
cellular accumulation of lipids, in particular
Figure 2. Cell densities and neutral:polar lipid ratio on replete and N-limited media a) Cell densities of C. pyrenoidosacultures grown in full medium and nitrogen limited medium. b) Relative proportions of neutral and polar lipids in C.pyrenoidosa cultures grown in replete medium. c) Relative proportions of neutral and polar lipids in C. pyrenoidosa culturesgrown in nitrogen-limited medium
26 BIOS
Volume 84, Number 1, 2013
neutral lipids. However, this study also shows
that while certain stress conditions, such as
nitrogen limitation, may increase the lipid
accumulation or the percentage of neutral lipids,
the total lipid productivity may be lower due to
the negative effect on growth. This study finds
that irradiance levels of 400 lE have a positive
effect on lipid productivity for C. pyrenoidosa
compared to 200 lE. We did not, however,
investigate higher irradiances. While higher
irradiance levels may have had an even greater
effect on lipid productivity, high irradiance
levels may also reduce algal growth rate through
photoinhibition (Radakovits et al., 2010) and
thereby reduce lipid productivity overall. These
results are also in agreement with previous
studies showing an increase in the proportion of
polar lipids with an increase in irradiance levels
(Gordillo et al., 1998). This can be explained by
the positive effect of a higher irradiance level on
growth. During a fast growth rate cells have to
produce polar lipids, such as phospho- and
sphingolipids, for cellular membranes while
storage lipids are mobilized to sustain a fast
growth rate.
Many studies have investigated the effect of
nitrogen limitation on lipid accumulation and
shown that during nitrogen starvation cells
increase total lipid accumulation, mainly in the
form of storage lipids (neutral lipids) (Converti
et al., 2009; Gordillo et al., 1998; Guschina and
Harwood 2006; Illman et al., 2000). Some
studies (Converti et al., 2009; Zhila et al., 2005)
have mentioned the negative effect of nitrogen
starvation on cellular growth but did not
correlate the effect of nitrogen limitation on
lipid productivity. The results of this study
suggest that nitrogen limitation has a negative
effect on lipid productivity if cultures are
limited for more than a week for the species
and conditions tested. As we only investigated
two levels of nitrogen, however, future studies
are planned to investigate a range of nitrogen
levels. This will help determine the most
optimal nutrient composition(s) that induce the
highest level of lipid accumulation without
excessively inhibiting growth, thus yielding
the greatest total lipid productivity
Carbon dioxide supplementation to algal
cultures is known to increase the growth rate
since the CO2 levels in aqueous environments
are very low. Previous studies have reported that
CO2 supplementation leads to an increase in
growth rate but no apparent increase in the
Figure 3. Cell densities and neutral:polar lipid ratio with 2 % CO2 sparging (C. pyrenoidosa) a) Cell densities of C.pyrenoidosa cultures grown with 2 % CO2. b) Relative proportions of neutral and polar lipids in C. pyrenoidosa culturesgrown with 2 % CO2.
Manipulation of algae for maximal oil production 27
Volume 84, Number 1, 2013
proportion of neutral lipids (Gordillo et al.,
1998, Chiu et al., 2009). Our results show the
same trend although prolonged supplementation
of CO2 led to a decrease of pH in the media
which probably led to the observed decrease in
growth. Based on the acquired data, a few days’
exposure to 2% CO2 or longer exposure to
lower levels of CO2 will increase lipid produc-
tivity by increasing cell count.
While the results of this are generally in
agreement with previous studies showing that
lipid accumulation is positively affected by
environmental stress, this stress-induced lipid
accumulation correlates negatively with total
lipid productivity due to a negative impact of
stress on growth. Our results stress the need for
future algal biodiesel research to focus more on
parameters increasing lipid productivity, not
only lipid accumulation, if biodiesel is to
become a viable option to gasoline.
Acknowledgements: Funding support for this
research was made possible through the Air
Force Office of Scientific Research and Air
Force Research Laboratories. The authors
would also like to thank Dr. Juergen Polle,
Brooklyn College, for supplying promising
algae strains.
The views expressed in this article are those of
the authors and do not reflect the official policy
or position of the United States Air Force, the
Department of Defense, or the US Government.
References
Banerjee, A., Sharma, R., Chisti, Y. and Banerjee, U. (2002).
Botryococcus braunii: A Renewable Source of Hydro-
carbons and Other Chemicals. Crit Rev Biotechnol. 22
(3), 245–279.
Bigogno, C., Khozin-Goldberg, I., Boussiba, S., Vonshak,
A., and Cohen, Z. (2002). Lipid and fatty acid
composition of the green oleaginous alga Parietochloris
incisa, the richest plant source of arachidonic acid.
Phytochemistry 60, 497–503.
Chisti, Y. (2007). Biodiesel from microalgae. Biotechnol
Adv. May-June, 25 (3), 294–306.
Chisti, Y. (2008). Biodiesel from microalgae beats bio-
ethanol. Trends Biotechnol. March, 26 (3), 126–131.
Chiu, S-Y., Kao, C-Y., Tsai, M-T., Ong, S-C., Chen, C-H.,
and Lin, C-S. (2009). Lipid accumulation and CO2
utilization of Nannochloropsis oculata in response to
CO2 aeration. Biores Technol, 100, 833–838.
Converti, A., Casazza, A.A., Ortiz, E.Y., Perego, P. and del
Borghi, M. (2009). Effect of temperature and nitrogen
concentration on the growth and lipid content of
Nannochloropsis oculata and Chlorella vulgaris for
Figure 4. Cell densities and neutral:polar lipid ratio with 2% CO2 sparging (Ettlia sp.) a) Cell densities of Ettlia sp.cultures grown with 2 % CO2. b) Relative proportions of neutral and polar lipids in Ettlia sp. cultures grown with 2 % CO2.
28 BIOS
Volume 84, Number 1, 2013
biodiesel production. Chem Eng Process, 48, 1146–1151.
Durrett, T.P., Benning, C., and Ohlrogge, J. (2008). Planttriacylglycerols as feedstocks for the production ofbiofuels. Plant J, May, 54 (4), 593–607.
Gordillo, F.J.C., Goutx, M., Figueroa, F.L. and Niell, F.X.(1998). Effects of light intensity, CO2 and nitrogensupply on lipid class composition of Dunaliella viridis. JAppl Phycol, 10, 135–144.
Greenwell, H.C., Laurens, L.M.L., Shields, R.J., Lovitt,R.W., and Flynn, K.J. (2010). Placing microalgae on thebiofuels priority list: a review of the technologicalchallenges. J R Soc Interface. 7, 703–726.
Guillard, R.R.L. and Ryther, J.H. (1962). Studies of marineplanktonic diatoms. I. Cyclotella nana Hustedt andDetonula confervaceae (Cleve). Can. J. Microbiol. 8,229–239.
Guschina, I. A and Harwood, J. L. (2006). Lipids and lipidmetabolism in eukaryotic algae. Prog Lipid Res, 45,160–186.
Hill, J., Nelson, E., Tilman, D., Polasky, S., and Tiffany, D.(2006). Environmental, economic, and energetic costsand benefits of biodiesel and ethanol biofuels. Proc NatlAcad Sci USA. July 25, 103(30), 206–10.
Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz,M., Seibert, M., and Darzins, A. (2008). Microalgaltriacylglycerols as feedstocks for biofuel production:perspectives and advances. Plant J, May, 54(4), 621–39.
Illman, A.M., Scragg, A.H. and Shales, S.W. (2000).Increase in Chlorella strains calorific values when grownin low nitrogen medium. Enzyme Microb Technol, 27,631–635.
Li, X., Hu, H.Y., and Zhang, Y.P. (2011). Growth and lipidaccumulation properties of a freshwater microalgaScenedesmus sp. under different cultivation temper-ature.Bioresour Technol. Feb;102(3), 3098–102
Lin, Q. and Lin, J. (2010). Effects of nitrogen source andconcentration on biomass and oil production of aScenedesmus rubescens like microalga. Bioresour Tech-nol. 102, 1615–1621.
Metzger, P. and Largeau, C. (2004). Botryococcus braunii: a
rich source for hydrocarbons and related ether lipids.
Appl Microbiol Biotechnol. February, 66 (5), 486–496.
National Renewable Energy Laboratory (NREL). Jet Fuel
from Microalgal Lipids. (Fact Sheet). 2006. 2 pp.; NREL
Report No. FS-840–40352.
Poyton, R.O. (1970). The Characterization of Hyalochlor-
ella marina gen. et sp.nov. a New Colourless Counter-
part of Chlorella. J Gen Microbiol. 62, 171–188.
Radakovits, R., Jinkerson, R.E., Darzins, A. and Posewitz,
M.C. (2010). Genetic engineering of algae for enhanced
biofuel production. Eukaryot Cell, 9, 486–501.
Rodolfi, L., Chini Zittellini, G., Bassi, N., Padovani, G.,
Biondi, N., Bonini, G. and Tredici, M. R. (2009).
Microalgae for oil: strain selection, induction of lipid
synthesis and outdoor mass cultivation in a low cost
photobioreactor. Biotechnol Bioeng, 102, 100–112
Roessler, P.G. (1990). Environmental control of glycerolipid
metabolism in microalgae: commercial implications and
future research directions. J Phycol. 26, 393–399.
Sheehan, J., Dunahay, T.G., Benemann, J.R., Roessler, P.G.,
and Weissman, J.C. (1998). A Look Back at the US
Department of Energy’s Aquatic Species Program:
Biodiesel from Algae - Close Out Report. 328 pp.;
NREL Report No. TP-580-24190.
Shelef, G., Sukenik, A., and Green, M. Microalgae
harvesting and processing: a literature review. Solar
Energy Research Institute, U.S. Department of Energy
Technical Report (SERI/STR-231-2396), August 1984.
Weldy C.S., and Huesemann M. (2007). Lipid production by
Dunaliella salina in batch culture: effects of nitrogen
limitation and light intensity. U.S. Department of Energy
Journal of Undergraduate Research. 7, 115–122.
Zhila N.O., Kalacheva, G.S. and Volova, T.G. (2005). Effect
of nitrogen limitation on the growth and lipid compo-
sition of the green alga Botryococcus braunii KutzIPPAS H-252. J Plant Physiol. 52(3), 311–319.
Received 18 December 2011; accepted 19 July 2012.
Manipulation of algae for maximal oil production 29
Volume 84, Number 1, 2013
top related