Direct Acid Methylation for Extraction of Fatty Acid

Content from Microalgae Cells

Benjamin D. Frigo-Vaz & Ping Wang
Received: 9 December 2013 / Accepted: 24 March 2014 /
Published online: 17 May 2014
#
Springer Science+Business Media New York 2014
Abstract Direct acid methylation was examined as a means for both analysis of fatty acid
content in microalgal cells and biodiesel production without pretreatment. Microalgal cells of
Chlamydomonas reinhardtii and Dunaliella tertiolecta were prepared and examined. It ap-
peared that direct acid methylation extracted higher fatty acid content than the solvent-based
Soxhlet extraction process. It also revealed that the latter was prone to extract a significant
amount of nonlipid hydrophobic impurities, including hydrophobic proteins and phytol-type
compounds, while direct methylation produces essentially pure ester product. This work
demonstrates that direct acid methylation provides superior fatty acid extraction, promising
an efficient process for either quantification of lipid content or production of biodiesel.
Keywords Fatty acid analysis
.
Lipid content
.
Algae biofuel
.
Acid methylation
.
Biodiesel
.
Lipid extraction
Introduction
Microalgae have been pursued extensively for production of fatty acids for production of
biofuels. This is because microalgae offer efficient photosynthetic conversion of CO
2
to
biomass [1, 2]. Most lipid contents are primarily determined via Soxhlet extraction or Nile
red dying, such as the processes suggested by US Department of Energys Aquatic Species
Program [3, 4]. These detection methods, though rapid, only provide a measure of lipid
quantity in terms of mass ratio with potential interferences from impurities of the biomass
and do not provide information about the composition of the resulted lipid extract. Fatty acids,
the most valuable components of algae for the purpose of biofuel production, generally
constitute only one type of the lipids in the extract detected or produced by these means.
Accurate and selective extraction of fatty acid of microalgal cells is not only important for the
purpose of quantification of valuable fatty acid content but also important for efficient
production of high-quality biofuel product.
Appl Biochem Biotechnol (2014) 173:15811586
DOI 10.1007/s12010-014-0881-7
B. D. Frigo-Vaz
:
P. Wang (*)
Department of Bioproducts and Biosystems Engineering, and Biotechnology Institute,
University of Minnesota, 2008 Folwell Ave., St. Paul, MN 55108, USA
e-mail: ping@umn.edu
Acid methylation has been shown as a reliable method for lipid extraction, which is
essentially the same reaction process for biodiesel production [5]. We can also expect that
acid methylation causes both caustic lysis of cells, and when conducted in the presence of
hydrophobic organic solvents, it simultaneously removes fatty acids from the lipid content of
the algal cells regardless of their location and existing chemical forms. The current work
therefore examines the potentials of acid methylation for extraction and conversion of fatty
acids to methyl esters without pretreatment of algae samples, in comparison to general
methods of solvent extraction.
Materials and Methods
Algae Preparation
Algae species tested in this work included Chlamydomonas reinhardtii and Dunaliella
tertiolecta. C. reinhardtii strain 137C was provided from the Chlamydomonas Resource
Center at University of Minnesota (250 Biological Sciences Center, 1445 Gortner Avenue,
St. Paul, MN 55108 USA), while D. tertiolecta was purchased from UTEX (205 W. 24th St.
Stop A6700 Austin, TX 787121240 USA). All cultures were grown under Verilux full-
spectrum fluorescent lighting with luminous flux determined to be greater than 3,000 lmbut no
greater than 8,500 lm. D. tertiolecta algae cultures had day/night cycles of 16/8 h provided by
timers on the fluorescent lighting. D. tertiolecta cultures were also fed carbon dioxide which
was bubbled through the culture media at flowrates no greater than 10 mL/s for 5 min each day.
D. tertiolecta was grown in sea salt saline growth media made from deionized water and
35 g/L of Morton Coarse Sea Salt. Growth media consisted of 1.2 mL/L P-IV trace metal
solution which contained 7.5 g/L of EDTA disodium salt, 0.97 g/L iron trichloride hexahy-
drate, 410 mg/L manganese chloride tetrahydrate, 50 mg/L of zinc chloride, 20 mg/L of cobalt
chloride hexahydrate, and 40 mg/L sodium molybdate dihydrate. Growth media also consisted
of 3.3 mL/L of 0.195 g/L sodium nitrate solution, 3.3 mL/L of 536 g/L disodium phosphate
hexahydrate solution, and 1 mL of vitamin B
12
solution containing 50 mM HEPES salt
adjusted to a pH of 7.8 and 135 mg/L vitamin B
12
.
C. reinhardtii growth media was made of 5 mL/L Beijerincks solution consisting of 100 g/
L of ammonium chloride, 4 g/L of magnesium sulfate heptahydrate, and 2 g/L of calcium
chloride dihydrate; 5 mL/L of phosphate buffer solution consisted of 288 g/L of dipotassium
phosphate and 144 g/L of monopotassium phosphate. C. reinhardtii growth media also
consisted of 5 mL/L P-IV trace metal solution.
After growth, algae cultures were harvested by centrifugation at 3,600 rpm (1,500 rcf) for
5 min, cleaned with Tris buffer, and brought to a final volume of 10 % algae pellet by volume.
C. reinhardtii algae samples were stored in Tris buffer, and D. tertiolecta was stored in Tris
buffer containing 3.5 % g/L sodium chloride. Multiple algae stocks were mixed together per
species to achieve a homogeneous single stock with uniform lipid content. This stock was
divided into ~45-mL ampules and refrozen at 20 C.
Soxhlet Extraction
Soxhlet extraction utilized 10 mL of either acetone or hexane following a procedure as reported
previously [6]. A Soxhlet reactor was operated via a water bath at 70 C, for 612 h, using
50100 mg of freeze-dried algae. Samples were vacuumdried and weighed and stored in sealed
containers at 4 C. Extract and residual material was weighed to determine extraction efficiency.
1582 Appl Biochem Biotechnol (2014) 173:15811586
Acid Methylation
Acid methylation solution consisted of 60 % methanol, 7 % acetyl chloride, and 33 %
tetrahydrofuran, similar to a procedure reported previously [7]. Samples of 1~5 mg were
exposed to 500 L acid methylation solution at 65 C for 2 h. Undecanoic acid (C11) was
added as an internal standard methylation reaction. Iso-octane was applied in 1:1 volume ratio
for extraction of ester product; samples were shaken at 200 rpm for 5 min, and then, saturated
sodium sulfate solution (250 L) was added to induce phase separation. Iso-octane phase was
retrieved as the top layer for gas chromatography (GC) analysis. Tridecanoate ester (C13) was
used as the internal GC analysis standard.
Protein Assay
Bicinchoninic acid (BCA) assay was chosen to measure free-floating protein concentrations in
extract samples because it is a very quick and simple protein assay protocol [8, 9].
Spectrometry was conducted with a Varian Bio50 UV-visible spectrophotometer. BCA
working reagent was made by combining 10 mL of Thermo Scientific Micro BCA
TM
(Thermo Scientific product # 23235) reagent A with 9.6 mL reagent B and 0.4 mL reagent
C. Soxhlet solvent extraction sample (1 mL) was vacuum dried first, diluted to 500 L with
water, and was then mixed with 500 L of BCAworking reagent. The mixture was heated in a
water bath for 60 min at 60 C; 50 L solution was removed and mixed with 450 L of
deionized water in a cuvette for spectrometry analysis (Abs 562 nm). A protein standard
solution of 2 mg/mL bovine serum albumin was used.
Chlorophyll Assay
A sample of 50 L of algae samples in Tris buffer was added to 1 mL of acetone and shaken
by hand before taking spectra. Spectrometry measurements were taken at 645 and 663 nm with
a blank Tris buffer solution as the reference. Chlorophyll concentrations and amounts were
determined for chlorophyll species A and B determined following a method as suggested
previously [10].
GC-FID and MS
For gas chromatography, a Varian 3900 equipped with a RTX-5 column (Shimadzu
Scientific Instruments, Inc., Columbia, MD) was used for flame ionization detection (FID).
AVarian Saturn 3 was used for mass spectrometry (MS). The analysis was conducted with
the injection of 1 L of sample with a split ratio of 10, into an injector chamber at 260 C. A
nitrogen gas was purged with a flow rate of 5 mL/min. Initial temperature for the column was
set to 165 C, ramped up at 1 C/min to 175 C. The column temperature was then raised up at
10 C/min to 225 C to clean the column before the next injection.
Results and Discussion
Solvent-based Soxhlet extraction process has been generally applied for assessment of lipid
content of algal cells. This method was first applied to the algae samples prepared in this work.
Two common solvents, acetone and hexane, were tested. It appeared that acetone extracted
much more content than hexane. For the tests with C. reinhardtii, content found in acetone
Appl Biochem Biotechnol (2014) 173:15811586 1583
extract was 221 % of the total cell mass (dry) tested, while hexane extracted only 62 %.
Extraction with acetone was then chosen for extraction tests with D. tertiolecta algae
samples, and it showed a result of 142 % of the total cell mass (dry). In comparison to
those results, direct acid methylation with algae samples (freeze-dried without further
pretreatment) showed moderate content of extraction, with 131 % determined for
C. reinhardtii and 51 % for D. tertiolecta. Acid methylation also appeared to be a quick
process, taking about 2 h at 50 C to complete (Fig. 1). Experimental results showed acid
methylation of freeze-dried C. reinhardtii algae to be completed in 1 h, further methylation
thereafter showing minor decline in fatty acid content which may be attributed to hydrolysis
effect of the ester product (Fig. 1).
Although acetone extraction generated better extraction as measured by mass ratio, it
appeared the extract contained impurities as indicated by its color and nonvolatile solid
residues. Solid residue was not found in the products of direct acid methylation, indicating
pure fatty acid ester product was recovered. Further GC analysis showed that the fatty acid
content recovered by direct acid methylation has a C16/C18 ratio of about 0.8 for
C. reinhardtii and 0.739 for D. tertiolecta.
Fig. 1 Reaction time course of acid methylation with C. reinhardtii samples. (Peak areas are represented as the
ratio to that of the C13 internal standard; diamond represents C18 and square C16)
Fig. 2 Composition of acetone Soxhlet extract content of C. reinhardtii and D. tertiolecta samples
(redprotein, greenchlorophyll, yellowfatty acid, grayunknown)
1584 Appl Biochem Biotechnol (2014) 173:15811586
With the above observation, we conducted acid methylation of the solvent extraction
samples, with an expectation to detect similar fatty acid content in the extract as that resulted
from direct acid methylation of the algal cells. As a result, it showed that the acetone extract of
C. reinhardtii contained 593 % fatty acids that can be recovered as methyl ester product,
equivalent to131 % of the algae cell mass (Fig. 2). The hexane extract, although low in terms
of overall yield, showed a bit higher fatty acid content, with 646 % identified as fatty acids.
D. tertiolecta algae Soxhlet acetone extract was found to contain only 365 % fatty acids
(over 60 % was nonfatty acid content), equivalent to 51 % of the cell mass (Fig. 1).
The remaining content of the solvent extraction appeared to be mostly chlorophyll and
proteins. Chlorophyll assays revealed that 8.30.6 % of D. tertiolecta acetone extract was
chlorophyll and that 174 % of C. reinhardtii acetone extracted was chlorophyll. BCA protein
assay results also showed that 5.31.2 % of D. tertiolecta acetone extract was protein while
that of C. reinhardtii was 144 %. The rest of the extracted content, mostly remained as solid
residues upon evaporation of the solvent, is unknown at this moment (Fig. 1).
When one compares the fatty acid recovery results, apparently direct acid methylation was
more efficient in recovering pure fatty acid content from the cell samples. Soxhlet tends to
extract large amounts of impurities from the biomass. The remaining cell residue after Soxhlet
extraction also appeared to contain additional fatty acid content when it was applied for acid
methylation tests. The residue of C. reinhardtii after acetone extraction was found to contain
2.40.3 % fatty acids (by mass). The sum of the fatty acid content in the cell residue and that
found in the solvent extract produced a total fatty acid content of 16.80.2 % for the algae
sample, which is in a close agreement found by direct acid methylation (~18 %). Tests with
Table 2 Concentration of C16 and
C18 (both saturated and unsaturat-
ed) fatty acids in D. tertiolecta
Tsuzuki et al. [12] Tsuzuki et al. [12] Volkman et al. [14] Current work
DT DT DT DT
Ambient CO
2
5%CO
2
N/A Daily CO
2
bubbling
0.23 0.242 0.147 0.190
0.27 0.258 0.288 0.211
0 0 0.004 0.015
0.49 0.483 0.523 0.584
0.50 0.5 0.435 0.425
0.49 0.483 0.527 0.575
1.03 1.04 0.83 0.739
Table 1 Concentration of C16 and C18 (both saturated and unsaturated) fatty acids in C. reinhardtii
Data source El-Sheekh [11] El-Sheekh 1993 Tsuzuki et al. [12] Tsuzuki et al. [12] Current work
Species CR CR CR CR CR
Growth conditions Autotrophy Heterotrophy Ambient CO
2
4 % CO
2
Ambient CO
2
C16 saturated 0.1626 0.173 0.266 0.382 0.22
C16 unsaturated 0.194 0.1762 0.203 0.101 0.23
C18 saturated 0.0922 0.0877 0 0 0.05
C18 unsaturated 0.4478 0.4602 0.5 0.501 0.51
C16 total 0.3566 0.3492 0.469 0.483 0.45
C18 total 0.54 0.5479 0.5 0.501 0.55
C16/C18 ratio 0.66 0.64 0.94 0.96 0.80
Appl Biochem Biotechnol (2014) 173:15811586 1585
residual D. tertiolecta algae samples showed less than 1 % fatty acids (by mass) in the residue
biomass with a total fatty acid content of 5~6 %. This observation further supported that direct
acid methylation could effectively recover fatty acid content from algae cells.
The distribution of fatty acid species found from solvent extraction and direct acid
methylation appears to be in agreement for both algae samples, with a total C16/C18 ratio
determined as 0.800.09 for C. reinhardtii (Table 1) and 0.7390.001 for D. tertiolecta
(Table 2). Those data generally agree with what had been reported previously in literature
but with a certain degree of variation (Tables 1 and 2). That may have been the results of
different culture conditions, as it has been generally accepted that fatty acid content could vary
greatly with growth conditions [1113]. C. reinhardtii and D. tertiolecta algae generally had
similar fatty acid compositions except in the case of saturated C18 (stearate), for which
D. tertiolecta algae had about a fourth as much as C. reinhardtii algae. Longer-chain fatty
acids beyond C18 such as C20 and C22, generally represented less than 12 % of the total
fatty acid content, may have been detected in the D. tertiolecta and C. reinhardtii samples
prepared in this work and accounted toward the C18 peak (no clearly separated peaks
observed).
Conclusion
The current work showed that direct acid methylation of algae provides superior advantages
over solvent extraction for both quantification of fatty acid content and production of biodiesel.
Solvent extraction tends to extract nonfatty acid components including chlorophyll and
hydrophobic proteins, representing significant percentages of the extracted content. Thus, lipid
content determined using Soxhlet may lead to overestimation of the potentials of algae cells for
biodiesel production. Direct acid methylation produces high-purity product and appears to be a
quick process that could be completed with 2 h, without pretreatment requirements.
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