Bioresource Technology 102 (2011) 94100

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Production of algae-based biodiesel using the continuous catalytic Mcgyan process

Brian J. Krohn, Clayton V. McNeff *, Bingwen Yan, Daniel Nowlan
SarTec Corporation, 617 Pierce Street, Anoka, MN 55303, USA

a r t i c l e

i n f o

Article history:
Received 25 February 2010
Received in revised form 11 May 2010
Accepted 17 May 2010
Available online 18 June 2010
Keywords:
Esterication
Transesterication
Algae
Biodiesel
Heterogeneous catalyst

a b s t r a c t
This study demonstrates the production of algal biodiesel from Dunaliella tertiolecta, Nannochloropsis oculata, wild freshwater microalgae, and macroalgae lipids using a highly efcient continuous catalytic process. The heterogeneous catalytic process uses supercritical methanol and porous titania microspheres in
a xed bed reactor to catalyze the simultaneous transesterication and esterication of triacylglycerides
and free fatty acids, respectively, to fatty acid methyl esters (biodiesel). Triacylglycerides and free fatty
acids were converted to alkyl esters with up to 85% efciency as measured by 300 MHz 1H NMR spectroscopy. The lipid composition of the different algae was studied gravimetrically and by gas chromatography. The analysis showed that even though total lipids comprised upwards of 19% of algal dry weight
the saponiable lipids, and resulting biodiesel, comprised only 1% of dry weight. Thus highlighting the
need to determine the triacylglyceride and free fatty acid content when considering microalgae for biodiesel production.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
In the last two decades growing international concern about the
negative environmental impacts of fossil energy has drawn significant attention to renewable liquid biofuels as a way to replace
petroleum-based fuels (Huntley and Redalje, 2007). Biodiesel, a
common term for long chain alkyl esters, is a renewable, biodegradable, and non-toxic biofuel that shows great promise. Biodiesel
is derived from the transesterication of mono-, di- and tri-acylglycerides (TAGs) and the esterication of free fatty acids (FFAs)
that occur naturally in biological lipids, such as animal fats and
plant oils. As a result, biodiesel has the potential to be a carbon
neutral fuel (Lopez et al., 2005; Ma and Hanna, 1999; Freedman
et al., 1984). Furthermore, in comparison to petroleum diesel, biodiesel emits lower levels of environmental pollutants including
volatile organic compounds, particulate matter, and sulfur-compounds during combustion (Swanson et al., 2007; Graboski and
McCormick, 1998; Schuchardt et al., 1998).
Although biodiesel itself has excellent fuel and combustion proles there are signicant concerns with its current production from
food crops, such as soybean, rapeseed, palm, or sunower oils
(Howarth and Bringezu, 2009). Recent studies have reported that
an increase in production of biofuels on arable land could lead to
an increase in deforestation, thereby releasing more CO2 into the
atmosphere than the biofuels would offset (Fargione et al., 2008;
Searchinger et al., 2008). To compound the problem, it has been
* Corresponding author. Tel.: +1 763 421 1072; fax: +1 763 421 2319.
E-mail address: claytonmcneff@sartec.com (C.V. McNeff).
0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.05.035

theorized that increased use of fertilizer to produce more biofuels

could release additional N2O from the soil and negate the effect of
reducing CO2 output from fossil fuels (Crutzen et al., 2008). Moreover, even if the production of biofuels from traditional crops was
increased dramatically in the US there simply is not enough farmland to meet the demand for fuel. For instance, it would require
594 Mha or 336% of existing farmland in the United States to produce enough biodiesel from soybeans, the main biodiesel feedstock
in the US, to replace only 50% of the transportation fuel consumed
in the US annually (Chisti, 2007). Finally, the cost of biodiesel is too
high to be economically viable without government supports due
to the high price of highly rened oils, which makes up 80% of
the cost of production (Haas et al., 2006; Hill et al., 2006; Demirbas,
2007). Thus, nding an inexpensive source of feedstock lipids that
does not use or displace farmland is imperative for biodiesel to
compete economically with petroleum diesel.
Since the mid 70s microalgae have been extensively researched
as a non-food based biodiesel feedstock and, due to their high
growth rates and oil content, show great promise as a feedstock
(Sheehan et al., 1998). Microalgae are estimated to produce biomass at a rate 50 times greater than the fastest growing terrestrial
plantswitchgrass (Li et al., 2008). In addition to high biomass productivity, microalgae can be comprised of 185% lipids by dry
weight (Chisti, 2007; Sheehan et al., 1998; Rodol et al., 2009;
Spoehr and Milner, 1949). In comparison to the 594 Mha needed
for soybeans, microalgae with 30% TAG or FFA content would only
require 4.5 Mha or 3% of existing US cropland to displace 50% of
USs transportation fuel with biodiesel (Chisti, 2007). Microalgaes
high growth rate and lipid content would make it possible to

B.J. Krohn et al. / Bioresource Technology 102 (2011) 94100

produce enough biodiesel to satisfy the enormous demand for fuels

in a relatively small area.
The benets of microalgal farming can be summarized thus: (1)
oil yield per acre can theoretically be 100 times higher than terrestrial oil crops (Chisti, 2007); (2) it does not compete with conventional agriculture for land resources because it can use brackish
water, saltwater, or wastewater on degraded land (Sheehan et al.,
1998); (3) even though it uses an aqueous environment, it consumes less water than terrestrial plants (Li et al., 2008); (4) the
recycling of biomass or using waste water reduces fertilizer use
and reduces N2O emissions (Sialve et al., 2009); (5) microalgae biomass production, needing 1.8 g of CO2 per 1 g of dry biomass, may
be coupled with the sequestration of waste CO2 (Doucha et al.,
2005; Doucha and Livansky, 1995); (6) a variety of products may
be produced from microalgae including other biofuels, animal feed,
and valuable pharmaceutical and cosmetic products (Huntley and
Redalje, 2007; Chisti, 2007; Li et al., 2008; Rodol et al., 2009;
Belarbi et al., 2000).
The majority of published research related to biodiesel production from algae has focused on increasing lipid productivity and
culturing algae on a large-scale, while there have been only a
few studies on the chemical conversion of microalgae lipids to biodiesel. Previous work describes the conversion of microalgae lipids
to alkyl esters via acid catalysis (Nagle and Lemke, 1990), or stepwise with the FFA esteried rst by acid catalysis and then the
remaining mixture of glycerides converted to fatty acid methyl esters (FAME) by base-catalyzed transesterication (Hossain et al.,
2008; Xu et al., 2006; Umdu et al., 2009; Zhu et al., 2008; Li
et al., 2007). Hossain et al. demonstrated the conversion of hexane
soluble lipid from Spirogyra sp. and Oedogonium sp. to biodiesel
with catalytic sodium hydroxide in methanol. Nagel and Lemke
tested both acid and base conversion of the total lipid fraction from
Chaetoceros muelleri and Monoraphidium minutum and obtained
yields of 68% and 32% with hydrochloric acid and sodium hydroxide catalysts, respectively. They speculated that the polar lipids and
high free fatty acid content in the total lipid fraction limited conversion. Xu et al. and Li et al. cultivated Chlorella protothecoides heterotrophically yielding a total lipid fraction high in saponiable
lipids and produced biodiesel using sulfuric acid and an immobilized lipase, respectively. Xu et al. achieved greater than 80% conversion and Li et al. claimed 98.15% conversion. Umdu et al. used
heterogeneous alumina supported magnesium oxide and calcium
oxide catalysts to convert 97.5% of the hexane soluble fraction in
Nannochloropsis oculata to biodiesel. While pyrolysis and liquefaction are more common methods for producing water soluble biooils from microalgae, the crude bio-oil produced is a complex mixture of compounds and is not technically biodiesel (alkyl esters)
that can be blended with petroleum diesel (Minowa et al., 1995;
Miao and Wu, 2006, 2004).
Here we report the use of a continuous heterogeneous catalyzed
process for the conversion of algal lipids to biodiesel. The conversion process (commercially known as the Mcgyan process) uses
ultra-stable porous metal oxide microspheres to simultaneously
esterify and transesterify FFA and TAG, respectively, under supercritical conditions (McNeff et al., 2008). Four different types of algae were carefully selected in this work to span the possible range
of algal lipids: Dunaliella tertiolecta is a salt water species and this
particular genus has already been cultivated on a commercial scale
(Lee, 1997). N. oculata, an eustugmatophyte, has recently been touted as the biodiesel algae because of its high lipid content and its
high neutral lipid productivity in an outdoor culture (Rodol et al.,
2009). A fresh water culture of Wild algae from a natural pond in
Minnesota was chosen because native wild algal species often
out compete mono-cultures in large-scale open pond systems. Finally, food-grade macroalgae lipid derived from Kelp was purchased (Bioengineering Inc., Hainan, China).

95

2. Experimental procedures and methods

2.1. Commercially available algae oil
Commercially rened macroalgae (Kelp) lipid was purchased
from Hai Nan Jin Yao Bioengineering Inc., Hainan, China. The Kelp
lipid provided sufcient volume for optimizing reactor conditions
and producing enough biodiesel under steady state conditions for
ASTM D6751 quality testing.

2.2. Algae growth monitoring

The Wild algae (Wild) and D. tertiolecta cultures were monitored daily by O.D. at 450 nm and chlorophyll a readings. Chlorophyll a content was measured with a Turner Designs Aquauor
handheld uorometer using the secondary standard PN 8000-950
to determine relative uorescent units (RFU). O.D. (x) was measured using a Milton Roy Spectronic 20D spectrometer and correlated to biomass concentration in g L1 (B) by a standard
curve, Eq. (1) for Wild and Eq. (2) for D. tertiolecta. Concentration
per unit was determined by dividing the total biomass in the
culture by the surface area of the culture vessels. The biomass
productivity (P) in g m2 day1 was determined by dividing the
difference between the rst (A1) and last (A2) areal biomass
readings in a growth period by the number of days in the
growth period (D), using Eq. (3). Biomass concentration in
g L1 was determined by ltering a known volume of culture
sample through pre-weighed 1.2 lm GFC Whatman lters and
drying to an ash free constant weight at 70 C and 500 torr.

B 0:23x 0:01R2 0:98

B 0:36x 0:08R2 0:98

1

P A2  A1 D

2.3. Algae biomass cultivation

2.3.1. Wild algae
SarTec Inc. (Anoka, MN) provided stock cultures of wild fresh
water algae native to Minnesota lakes and ponds. The stock cultures were created by taking 1 L water samples from Minnesota
lakes and ponds and then propagating the algae in the laboratory
in a modied Bolds media, noted below. The stock cultures were
cultivated under articial light (100200 lmol m2 s1), bubbled
with supplemental CO2, and maintained from 2007 to 2009 in
200 L drums. Under the cultivation conditions the Wild algae populations within the mixed culture reached a stable equilibrium,
which consisted predominately of Chlorella sp. with Scenedesmus
sp. and Chlamydomonas sp. making up a small percent of the
population.
The Wild algae were cultivated outside from May to early June
2009 (Andover, MN lat.: 45.2663; lon.: 93.3666) in two high density polyethylene totes (91 cm  114 cm  91 cm) with a culture
depth of 70 cm to 73 cm (750 L). The totes were inoculated with
Wild algae to obtain a starting concentration of 0.0100 g L1 based
on O.D. A mixture of 5% CO2 and air was bubbled through the culture
at 200300 mL min1; CO2 ow was turned off for 9 h over night. The
average pH and temperature were 7.6 (r = 0.5) and 18.1 C (r = 3.3),
respectively. The basal media was a modied Bolds formula (Barsanti and Gualtieri, 2006): (mg/L H2O): KH2PO4 (175), CaCl2 (19),
MgSO47H2O (75), NaNO3 (250), K2HPO4 (75), NaCl (25), H3BO3
(10.9), Na2EDTA (10), KOH (6.2), FeCl36H2O (4.8), MnCl44H2O
(1.8), ZnSO47H2O (0.22), NaMoO45H2O (0.39), CuSO45H2O (0.08),
Co(NO3)26H2O (0.05), Biotin (0.5), B12 (0.5), Thiamine B1 (100).

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B.J. Krohn et al. / Bioresource Technology 102 (2011) 94100

2.3.2. Dunaliella tertiolecta

Stock cultures of D. Tertiolecta were generously provided by Dr.
Arun Goyal (Anoka-Ramsey Community College, Coon Rapids, MN).
The stock cultures volume was doubled every 34 days by adding
fresh media. As their volume increased the cultures progressed
from 250 mL Erlenmeyer asks, to 2 L asks, to 40 L clear plastic
tubs, and nally 100 L clear plastic tubs. The stock cultures were
grown under articial lights (4060 lmol m2 s1) and bubbled
with a mixture of 5% CO2 and air.
Dunaliella tertiolecta cultures were cultivated outside from June
to July 2009 (Andover, MN lat.: 45.2663; lon.: 93.3666) in 200 L
drums and high density polyethylene totes (91 cm  114 cm 
91 cm). Stock cultures of D. tertiolecta were used to inoculate
180 L of media in 200 L drums to obtain a cell density of
0.1000 g L1 based on O.D. Once a cultures cell density had tripled to 0.3000 g L1 it was transferred to a tote and incrementally
diluted to 350 L. In both types of vessels a mixture of 10% CO2 and
air was bubbled through the culture at 200300 mL/min to
maintain a pH average of 6.8 (r = 0.5); CO2 ow was turned off
for 9 h over night. The temperature of the cultures averaged
20.7 C (r = 3.1). The basal media was a 1% salt media: (mg/L
H2O): MgCl26H2O (1500), NaCl (10,000), MgSO47H2O (500), KCl
(200), CaCl22H2O (260), KNO3 (500), KH2PO4 (50), NaCl (10,000),
FeCl3 (3), EDTANa (1.6), ZnCl2 (0.04), H3BO3 (0.6), CoCl26H2O
(0.0142), CuCl22H2O (0.04), MnCl24H2O (0.4), (NH4)6 Mo7O24
4H2O (0.38).
2.3.3. Nannochloropsis oculata
High density cultures of N. oculata were purchased from AlgaGen LLC (Vero Beach, FL). N. oculata was grown in an open pond,
with natural light, at pH of 8.0 and temperature of 29.4 C. An f/2
nutrient recipe in natural seawater that was carbon ltered, UV
treated, ozonated, and chlorinated comprised the basal media
(Barsanti and Gualtieri, 2006; Stenn; 2009): (mg/L H2O): NaNO3
(75), NaH2PO4H2O (5), FeCl36H2O (3.25), Na2EDTA (4.16),
MnCl44H2O (0.18), Co(NO3)26H2O (0.01), ZnSO47H2O (0.022),
NaMoO45H2O (0.006), Biotin (0.5), B12 (0.5), Thiamine B1 (100).
2.4. Harvesting
Algae biomass was harvested using a continuous feed, xedbowl centrifuge. The resulting paste, consisting of 1520% dry matter, was immediately frozen. The paste was then dried in a vacuum
oven at 70 C and 500 torr and stored at 15 C.
2.5. Extraction of lipid from microalgae
The neutral lipids that were converted to FAME using the xed
bed reactor were extracted from dried algae powder using hexane
Soxhlet extraction. Prior to the extraction dried algae akes were
pulverized using a blender and then ground in a ball mill for
5 days. Once the cells had been nely ground, they were placed
in the Soxhlet extractor until the extracting hexane was colorless
(2448 h). The lipid containing hexane solution was then ltered
through a 1.2 lm GFC Whatman lter. The hexane was then removed using a rotary evaporator leaving the black colored hexane
soluble lipids, which were labeled crude oil. The crude oil was
then re-dissolved in hexane and ltered through activated carbon
to remove pigments. The resulting neutral lipids were transparent
and ranged from light yellow to amber in color.
2.6. Gravimetric analysis of lipids
The total lipid content, hexane soluble lipid crude oil content,
and neutral lipid content was determined gravimetrically using a
modied Bligh and Dyer extraction method (Bligh and Dyer,

1959; Zhukova and Aizdaicher, 1995; Volkman et al., 1989; Mansour et al., 2005). The moisture content of wet algae paste was
determined using a Mettler-Toledo HR83 halogen moisture-analyzer (Columbus, OH). The moisture content in the samples was
then used to determine the necessary initial volume of chloroform
and methanol for the following extraction. Approximately 5 g of
wet algae paste was vigorously stirred in a solution of chloroformmethanolwater (1:2:0.4 v/v/v) for 1 h. Chloroform was
added resulting in a volume ratio of 1:1:0.4 (CHCl3:MeOH:H2O v/
v/v) and stirred for 20 min followed by the addition of deionized
water (1:1:0.8 CHCl3:MeOH:H2O v/v/v) and another 20 min of stirring. The extract was then ltered through a 1.2 lm GFC Whatman
lter and the residue extracted 4 times with 25 mL of chloroform
methanol (2:1) resulting in the removal of color from the algal
paste. Additional methanol and water was added to the extract
to return the volume ratio to 1:1:0.8 and separated in a separatory
funnel0.5 g of NaCl was added to break the emulsion. The organic
layer was removed and the aqueous phase was washed 3 times
with 10 mL aliquots of chloroform. The organic layer and aliquots
were combined and then dried with sodium sulfate, ltered
through a 1.2 lm GFC Whatman lter, and the volatiles were removed using a rotary evaporator. The resulting lipid fraction was
weighed and designated as the total lipids. The total lipids were
then extracted with hexane and similarly ltered and dried to yield
the crude lipids. Finally, the crude lipids were re-dissolved in hexane and ltered through activated carbon to yield the neutral lipid
fraction. Extractions for each algal culture were performed in quadruplicate and the mass percentage values averaged.
2.7. Conversion of algal lipids to biodiesel using a xed bed reactor
Preparation of the reactor and porous titania catalyst were previously described (McNeff et al., 2008). The previously described
method, however, was designed to process several liters of oil
and needed to be modied to accommodate the small volume of
algal lipids available. As a result, instead of using two separate
reactant streams of methanol and lipids, the modied system used
a single-reactant stream. Hexane was used as a carrier solvent to
create a single homogeneous feedstock solution. The feedstock
solution was comprised of 12 g of algae lipids, methanol and hexane (1:3:96 lipids:MeOH:hexanes w/w/w). Also, the previously described EFAR system was not employed.
The system was ushed with 1 L of methanolhexane solution
(3:97 MeOH:hexanes w/w) before and after each sample and the
feedstock ask was continuously sparged with nitrogen. One high
pressure Waters 560 HPLC pump (Milford, MA) was employed to
rst pump the solution through an empty stainless steel reactor
(1 cm i.d.  15 cm long) tted with 2 lm stainless steel frits to lter the stream before it entered the reactor. Then the reactant
stream passed through a heat exchanger where it exchanged heat
with the hot efuent leaving the reactor. Prior to passing through
the reactor, the reactant stream was brought up to temperature
by an independently controlled electrically driven preheater. The
backpressure of the system was maintained through the use of a
backpressure regulator obtained from Tescon (Elk River, MN). Once
the preheater and rector stabilized at 340 C with 2250 psi front
pressure and backpressure, the reactants were pumped across
the reactor with a 30 s residence time.
2.8. Conversion of Kelp lipids for ASTM testing
The modied process in Section 2.7 was not employed in converting Kelp oil for the ASTM testing. Instead, 2 L of commercially
purchased Kelp oil were converted to FAMEs using the Mcgyan
process and the easy fatty acid removal (EFAR) system previously
described by McNeff et al. Optimum conditions for the conversion

B.J. Krohn et al. / Bioresource Technology 102 (2011) 94100

of Kelp oil to biodiesel in the two reactant stream method were

360 C, 2295 psi, 30 s residence time, and a 32:1 MeOH:oil ratio.
The methanol was removed from the crude biodiesel by rotary
evaporation under reduced pressure and the distilled biodiesel
was transferred to a separatory funnel where the remaining water
was removed. The remaining FFAs in the biodiesel were removed
using the EFAR process previously described (McNeff et al.,
2008). After distillation of the remaining methanol the resulting
biodiesel was sent to FuelOnly Inc. (Vancouver, WA) for ASTM testing. For comparison of ASTM results, soybean biodiesel was produced and tested using the same methods.

97

tube containing 1 mL saturated NaHCO3 and 2 mL pentane. The

two layers were agitated by pipette until gas evolution ceased.
The pentane solution was carefully decanted by pipette and transferred to another test tube and dried over 4 molecular sieves. The
pentane was evaporated by drying with a stream of air and the
resulting residue was taken up in 1 mL of HPLC grade methanol
and analyzed by GCFID and GCMS.
2.12. Gas chromatographymass spectrometry (GCMS)

The conversion of neutral lipids to FAMEs was calculated using

a Varian 300 MHz 1H NMR (Palo Alto, CA) (McNeff et al., 2008).
Samples were prepared in CDCl3. The percent conversion was calculated by the ratio of the area of the singlet peak associated with
methyl esters at d 3.67 ppm and the peaks at d 2.2 ppm representative of the a-methylene protons (Knothe, 2001). The spectra for
the lipid samples were recorded on a Varian 500 MHz 1H NMR,
and the TAG content was analyzed using the method described
by Satyarthi et al. (2009)

An Agilent 6890 gas chromatograph electron impact mass spectrometer was also used to analyze the FAMEs in the algal biodiesel.
One microliters of sample, described in Section 2.11, was injected
in splitless mode at a ow rate of 1.0 mL/min with helium as the
carrier gas onto a (5% phenyl)-methylpolysiloxane column (DB-5;
30 m  0.25 mm i.d.; 0.25 lm lm thickness). The elution temperature program had an initial temperature of 50 C and then linearly
ramped to 180 C at 15 C min1, then to 230 C at 2 C min1, and
nally to 310 C at 30 C min1. The nal temperature was held for
13.67 min (total run time = 50 min). Mass spectra were acquired
using HP6890 MS software and peaks identication was aided with
the NIST MS library. The observed mass range was set from 37 to
800 amu to remove the solvent.

2.10. Gas chromatography (GC) analysis of FAMEs

2.13. Acid number method

Gas chromatography was used to determine the composition of

the different chain length FAMEs in the biodiesel products. GC
analysis was performed on an Agilent 6890 gas chromatograph tted with an HP 6890 autosampler using ame-ionization detection
(FID). Injector and detector temperatures were set at 240 and
325 C, respectively. Samples were dissolved in hexane containing
methyl heptadecanoate as an internal standard. One microliters
was injected in split mode (25:1) at a ow rate of 1.4 mL/min with
helium as the carrier gas onto a (5% phenyl)-methylpolysiloxane
column (HP-5; 30 m  0.032 mm i.d.; 0.10 lm lm thickness).
The elution temperature program had an initial temperature of
50 C and then linearly ramp to 180 C at 15 C min1, then to
230 C at 2 C min1, and nally to 250 C at 10 C min1. The nal
temperature was then held for 15.33 min (total run time = 51 min).
Individual peaks were identied using a combination of GCMS
and comparison of Rts to standards of fatty acid methyl esters
(FA: C12:0, C14:0, C15:0, C16:0, C18:0, C18:1(n  9), 18:2(n  6),
C18:3(n  3) (SigmaAldrich, St. Lois, MO).
Eqs. (4) and (5) were used to determine the percent biodiesel
conversion (%FA) by summation of the individual FAMEs in the
converted samples, where MFA is the mass of the fatty acid of interest, Misd is the internal standard mass, Ms is the mass of the sample,
AFA is the peak area of the fatty acid of interest, and Aisd is the peak
area of the internal standard. Coefcients a and b were determined
using a linear standard curve (R2 = 0.99). Peak areas were integrated using the HP 6890 GC software.

The acid number was determined using a Metrohm 839

Titrando titrator, 803 Ti Stand, 800 Dosino 10 mL dispensing
system, and 014 pH Unitrode glass electrode (Riverview, FL).
Samples were prepared in 110 mL solution of water, isopropyl
alcohol, and toluene (1:10:100 v/v/v) and titrated with 0.1 N
KOH in isopropyl alcohol.

2.9. NMR methods



AFA
a
b
Aisd

 
1
M FA
Ms
%FA

Misd
M isd
MFA
M isd

4
5

2.11. HCl catalyzed methanolysis of algal lipids for GCMS analysis

Approximately 45 mg of algal lipid was added to a small Teon
capped vial (2 mL) and 0.2 mL methanol were added. A solution of
0.8 mL of 5% acetyl chloride in methanol was added to the vial. The
vial was tightly capped and heated in a sand bath at 60 C for 6 h.
After cooling, the contents of the vial were transferred to a test

3. Results and discussion

3.1. Algae productivity
The biomass productivity of the outdoor cultures of Wild and D.
tertiolecta was 3.75 and 2.77 g m2 d1, respectively. Open-outdoor
systems using similar cultures have reported an average areal productivity of 1020 g m2 d1 (Lee, 2001). While the culture system
used in this study produced biomass at a lower rate, it did provide
sufcient lipids for further use in biodiesel production studies.
3.2. Gravimetric analysis of lipid content
The percent lipid in dry biomass was determined gravimetrically by chloroform/methanol extraction. Three different classes
of lipid were analyzed: total lipids, crude lipids, and neutral lipids.
The total lipid fraction includes: pigments, phospholipids, glycolipids, in addition to the neutral lipids (Hu et al., 2008; Gordillo et al.,
1998). The crude lipid fraction contains all neutral lipids and pigments. The neutral lipid fraction includes: TAGs, FFAs, hydrocarbons, sterols, wax and sterol esters, and free alcohols. The lipid
composition of microalgae is shown in Fig. 1. The neutral lipids
comprised 4.5 0.2%, 5.6 1.0%, and 9.0 2.0% of the Wild, D. tertiolecta, and N. oculata biomass, respectively, while the total lipids
made up 15.8 3.1%, 19.0 0.8%, and 18.0 3.5%, respectively.
Neutral lipids only comprised about 30% of total lipids for Wild,
D. tertiolecta, and 50% for N. oculata. Volkman et al. and Vanitha
et al. also reported that neutral lipids were about 35% of total lipids
for a wide variety of algal species.
The intense interest in microalgae for biodiesel stems primarily
from microalgaes high lipid content and high potential biomass
productivity per unit surface area compared to traditional crops.

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B.J. Krohn et al. / Bioresource Technology 102 (2011) 94100

Table 1
The acid numbers of the algal lipid feedstocks and the percent conversion of FFAs and
TAGs to FAMEs measured by 1H NMR and percent FAMEs in a sample measured by gas
chromatography after conversion using the single-reactant stream Mcgyan process
(Section 2.7).
Species

Kelp
Wild
D. tertiolecta
D. tertiolecta
(crude)
N. oculata

Fig. 1. Gravimetric analysis of the lipid content of Wild algae, D. tertiolecta, and N.
oculata showing total lipid, crude lipid, and neutral lipid composition.

However, the algal biodiesel productivity is often confused with total lipid productivity. For pure ASTM grade biodiesel, only the FFA
and TAG the content rather than the total lipid content must be
considered for biodiesel production potential. Fig. 1 shows that
the neutral lipid fraction and therefore the FFA and TAG available
to produce biodiesel makes up only a small fraction of the total lipids. It is possible to achieve high total and neutral lipid content by
using environmental stresses such as nutrient limitation, irradiance, temperature, or pH (Hu et al., 2008; Hu, 2004). Environmental stresses, however, have dramatically differing effects depending
on the algal species. For example, two commercially grown green
algae, Chlorella sp. and Dunaliella sp., show opposite responses under nitrogen deprivation. Chlorella can accumulate total lipids up to
85% dry weight (Spoehr and Milner, 1949) while Dunaliellas total
lipid content decreases (Gordillo et al., 1998; Borowitzka, 1999).
A combination of the right culture conditions and species is therefore required to achieve high levels lipid content. In selecting conditions and species for biodiesel production, however, the TAG and
FFA content will be more important than total lipids for nal biodiesel production.
Furthermore, the rate at which TAG and FFA are produced by
the algae may be a more important factor than simply total lipid
content. Stress induced lipid production does have a downside in
that the majority of algae species high lipid content is obtainable
only by sacricing biomass production, thus causing a net reduction in lipid productivity (Sheehan et al., 1998). In 2008, Rodol
et al. demonstrated for the rst time an enhancement of both
TAG content and TAG productivity in an outdoor culture by growing N. oculata under nitrogen deprivation and high irradiances. We
are thus in agreement with Rodol et al. whom have emphasized
the importance of considering TAG and FFA productivity and not
just percent total lipid content when selecting microalgae for
biodiesel.

3.3. Continuous conversion of algal lipid to FAME using microporous

titania catalyst under supercritical alcohol conditions
Table 1 shows the percent conversion of microalgae and rened macroalgae FFA and TAG to FAME, calculated by
300 MHz 1H NMR, using the single-reactant stream method
described in Section 2.7. The conversion of D. tertiolecta and
Wild algae FFA and TAGs to FAMEs was found to be between
80% and 85%. FFAs and TAGs from N. oculata and the crude lipids

Acid number
(mg KOH)

34.9172
102.5237
167.2505
117.7146
83.4012

H NMR

GC

Comments

FFA and TAG

Conversion
(%)

FAME in
sample (%)

No TAG
No TAG
No TAG
Other artifacts
no TAG
Other artifacts
no TAG

85.5
84.4
82.3
69.9

94.7
31.0
20.9
19.0

11.4

3.3

from D. tertiolecta had articially lower conversions as calculated

by this method due peak broadening caused by other biological
constituents resonances overlapping with the a-methylene proton peaks. The 1H NMR spectrum of biodiesel from Wild algae
is very similar to the 1H NMR of biodiesel from virgin soybean
oil (McNeff et al., 2008; Knothe, 2001).
In order to determine the FAME content of the processed lipids,
gas chromatography was used to quantify the percent FAMEs by
mass in the products. The FAME content, as determined by GC
FID, for the microalgae samples was found to be low, ranging from
3% to 31%, indicating that the bulk of the sample was comprised of
unsaponiable material. Low FAME content, however, is not unexpected; Vanitha et al. reported that under various light conditions
the FFAs and TAGs from Dunaliella viridis comprised only 3852% of
the neutral lipid fraction. The remaining neutral lipids were hydrocarbons, wax and sterol esters, free alcohols, and sterols. It is unclear how large percentages of these compounds would affect the
fuel properties of algal biodiesel. The highly rened Kelp oil yielded
94.7% FAME due to the fact that it is comprised entirely of FFAs and
TAGs. Table 2 shows the percent total lipids, neutral lipids, and
FAMEs to biomass, with FAMEs only making up 1% of biomass
compared to 1519% total lipids. This further illustrates the importance of high TAG and FFA content for microalgae to be a viable
source of biodiesel production.
The fatty acid composition was predominately C16 and C18
methyl esters, combined making up 85% to 100% of the fatty acids
in the microalgal lipids. Traditional biodiesel crops (soybean, corn,
rapeseed, and palm) are also dominated by C16 and C18 fatty acids.
The fatty acid composition relative to total fatty acid content is
shown in Table 3. The unsaturated C18 derivatives C18:1, C18:2,
and C18:3 were analyzed together due to co-elution. Interestingly,
the Kelp lipids contained shorter fatty acids with C12:0 making up
the largest fraction at 37.0%. These shorter chain FAME resulted in
a very high cetane number of 71.67, see Table 5.
1
H NMR also revealed that the D. tertiolecta, N. oculata and Wild
algae lipid fractions contained no measurable amount of TAGs.
Table 1 shows that the neutral lipids also have high acid numbers
(indicating that the neutral lipid fractions have high FFAs content)
and no TAG (determined by 1H NMR). An acid number less than

Table 2
Total lipid, neutral lipid, and FAME content of biomass dry weight of the three algal
cultures: Wild, D. tertiolecta, and N. oculata.
Composition of algal biomass (%)

Total lipid
Neutral lipid
FAME

Wild

D. tertiolecta

N. oculata

15.8
4.5
1.4

19.0
5.6
1.2

18.0
9.0
0.3

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B.J. Krohn et al. / Bioresource Technology 102 (2011) 94100

Table 3
Fatty acid composition of Kelp, Wild, D. tertiolecta, N. oculata and D. tertiolecta crude
lipids (relative mass %).
Fatty acid composition (relative mass %)
Kelp

Wild

D. tertiolecta

D. tertiolecta (crude)

N. oculata

C12:0
C14:0
C15:0
C16:0
C18:1,2,3
C18:0

51.1
14.0

6.6
25.7
2.6

47.1
38.3
7.0

44.3
47.9
7.8

40.9
44.1
14.9

100.0

Total

100.0

100.0

100.0

100.0

100.0

7.5

200 mg KOH (what it would be for pure FFA) and no TAGs indicate
that there are other unsaponiable compounds present in the neutral lipids. The high FFA content may be an artifact of TAG degradation during the extraction process but other studies also indicate
that algae typically contain a high ratio of FFA to TAG (Volkman
et al., 1989; Mansour et al., 2005; Gordillo et al., 1998; Vanitha
et al., 2007).
The high FFA content of microalgal lipid is a major topic that
must be addressed when considering an algal biodiesel process.
The conversion efciency of the traditional methods for biodiesel
production, homogeneous acid or base-catalyzed (e.g. H2SO4 or
KOH) transesterication of TAGs, is highly dependent on the FFA
and water content in the lipid feedstock (Lopez et al., 2005; Freedman et al., 1984; Kusdiana and Saka, 2004). The base catalyzed
method, used in the majority of current commercial operations,
cannot tolerate FFA content higher than 0.6% without unwanted
byproduct formation or drastically lower conversion efciency
(Ma and Hanna, 1999). This extreme sensitivity to even moderate
FFA content has proven a major hurdle for commercial biodiesel
production because inexpensive feedstocks, such as used vegetable
oils, tallow, and other waste oils, typically have high FFA and water
content (Ma and Hanna, 1999; Kusdiana and Saka, 2004). The traditional methods for converting oils with high FFA content are not
considered cost effective on a large-scale and as a result the traditional base catalyzed method is limited to the use of expensive
low-FFA content vegetable oils such as virgin soybean oil (Kasteren
and Nisworo, 2007). Since microalgal lipids contain very high levels of FFAs and currently cost at best $5/L (Rodol et al., 2009),
additional FFA purication or stepwise conversion of the lipids is
likely to be too expensive to be an economically viable solution.
Therefore, we believe an efcient method to simultaneously convert the TAGs and FFAs in microalgae to biodiesel is essential for
the future of algal biodiesel.

3.4. ASTM tested algal biodiesel

The continuous Mcgyan process has many benets over the
conventional base catalyzed process as summarized in Table 4. In
September of 2009 a 3 million gallon per year biodiesel plant
utilizing the Mcgyan process came online in Isanti, MN. Thus
demonstrating the scalability and economic viability of a continuous biodiesel process catalyzed by titania and supercritical alcohol.
Also, the Mcgyan process is ideally suited to convert microalgae
oil to biodiesel because of its tolerance of elevated levels of FFAs
and its exibility towards varying feedstock composition, as demonstrated in this paper. Moreover, as demonstrated in McNeff et al.,
the catalyst does not degrade over time allowing for long-term use
and recycling of the catalyst. Using the conventional base catalyzed
biodiesel processes the microalgae lipids would have to be puried, which would signicantly increase the cost of microalgae biodiesel production (Haas et al.).

Table 4
A comparison of the supercritical xedbed continuous ow process (the Mcgyan
process) to the conventional homogeneously base catalyzed batch system (typically
KOH or NaOH).

Consumes catalyst
Uses large amounts of H2O
Produces waste products
Produces soap byproducts
Produces glycerol as byproduct
Requires large footprint
Sensitive to H2O
Sensitive to FFA
Large quantities of strong acid/
base
Conversion rate
Can use a variety of feedstocks
Is a continuous process

Mcgyan
process

Homogeneous
process

No
No
No
No
No
No
No
No
No

Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes

Sec.
Yes
Yes

Hrs.Days
No
No

Table 5
ASTM testing, conducted by FuelOnly Inc., of macroalgae (Kelp) biodiesel and soybean
biodiesel.

a
b

Test

Algal
biodiesel

Soybean
biodiesela

ASTM
limits

ASTM
method

Flash point, PMCC

Distillation, 90%
recoveryb
Carbon residue

149
371

122
339

93 C min
360 C max

D93
D1160

0.018

0.019

D4530

Total glycerin

0.169

0.161

Free glycerin

0.006

0.005

Water and sediment

<0.005

3.07

Sulfur, total
Cetane number
Cloud point C
Sulfated ash
Copper strip corrosion
Acid number
Kinematic viscosity
at 40 Cb
Cold soak ltration
Phosphorus

8.43
71.67
16
0.008
1
0.01
9.8

3.07
50
+2
0.011
1
0.07
4.5

0.050
(% mass) max
0.240
(% mass) max
0.02
(% mass) max
0.05
(% volume)
max
15 ppm max
47 minimum

126
<1

185
2

D6584
D6584
D2709

0.020 max
No. 3 max
0.50 mg KOH/g
1.96.0 cst

D5453
D613
D2500
D874
D130
D664
D445

360 s max
10 ppm max

D6217b
D4951

Soybean biodiesel produced from the Mcgyan process.

Tests that did not meet ASTM standards.

The rapid conversion of high FFA content algal lipids to highgrade biodiesel was demonstrated using Kelp lipids and the twostream Mcgyan process, described in Section 2.8. The two-stream
method is similar to the commercialized process but it could only
be applied to the Kelp lipids because it requires several liters of oil.
Using the two-stream system the Kelp TAG and FFA were converted to FAME with 93.3% efciency as measured by 1H NMR.
Interestingly, the two-stream system had a higher percent conversion of TAG and FFA than the single-reactant stream system. The
lower conversion in the single-reactant stream may be due to
interference from the carrier solvent (hexane) in the reaction process. The two-stream Mcgyan process had similar yields to Umdu
et al. who reported 97% conversion of N. oculata lipids to biodiesel
by using CaO/Al2O3 as a heterogeneous catalyst in a batch system.
The use of supercritical conditions, titania microspheres, and a
continuous process in our study, however, resulted in a much faster reaction time of 30 s instead of 4 h. Finally, the Kelp biodiesel
passed all but two ASTM D6751 tests, distillation 90% recovery
and kinematic viscosity at 40 C as shown in Table 5, both ex-

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B.J. Krohn et al. / Bioresource Technology 102 (2011) 94100

ceeded the designated range. The ash point and cetane number
were also high compared to soybean based biodiesel while the
cloud point was notably lower at 16 C.

4. Conclusion
The major hurdle immediately facing microalgal biodiesel is the
large-scale cultivation of an algal species with high FFA and TAG
productivity. However, if sufcient quantities of microalgal lipids
can be cultured, harvested, and extracted the nal hurdle will be
the conversion lipids to FAME. Algal lipids, however, have high
FFA content, which will limit its use in the traditional biodiesel
process. We address this issue by demonstrating the conversion
of algal lipids to FAME using a continuous ow system catalyzed
by high temperature, high pressure, and titainia microspheres in
a xed bed reactor.

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