U.S. patent application number 14/804962 was filed with the patent office on 2015-11-12 for direct transesterification of algal biomass for synthesis of fatty acid ethyl esters (faee).
The applicant listed for this patent is HELIAE DEVELOPMENT LLC. Invention is credited to Sandip SHINDE.
Application Number | 20150322378 14/804962 |
Document ID | / |
Family ID | 50687640 |
Filed Date | 2015-11-12 |
United States Patent
Application |
20150322378 |
Kind Code |
A1 |
SHINDE; Sandip |
November 12, 2015 |
DIRECT TRANSESTERIFICATION OF ALGAL BIOMASS FOR SYNTHESIS OF FATTY
ACID ETHYL ESTERS (FAEE)
Abstract
Methods of producing fatty acid ethyl esters (FAEE) using a
direct transesterification process are described. The direct
transesterification process uses low levels of chemical solvents,
acid catalysts, and heating energy to produce the FAEE in a method
with increased efficiency in a co-solvent system. The FAEE produced
may be used in a variety of products including health, beauty,
nutraceutical, and cosmetic products.
Inventors: |
SHINDE; Sandip; (Gilbert,
AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HELIAE DEVELOPMENT LLC |
Gilbert |
AZ |
US |
|
|
Family ID: |
50687640 |
Appl. No.: |
14/804962 |
Filed: |
July 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2014/027161 |
Mar 14, 2014 |
|
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14804962 |
|
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61798436 |
Mar 15, 2013 |
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Current U.S.
Class: |
554/167 |
Current CPC
Class: |
C11C 1/10 20130101; C11C
1/08 20130101; C11C 3/10 20130101; C11C 1/007 20130101; C11B 3/04
20130101; C11B 3/12 20130101; C11C 1/005 20130101 |
International
Class: |
C11C 3/10 20060101
C11C003/10; C11C 1/00 20060101 C11C001/00; C11C 1/08 20060101
C11C001/08 |
Claims
1. A method for converting lipids in a lipid containing biological
material into fatty acid ethyl esters (FAEE), comprising: a. mixing
biological material comprising lipids and biomass with a first
non-polar solvent at a biomass:first non-polar solvent ratio of 1:1
to 1:10 to form a first reaction mixture; b. mixing the first
reaction mixture with ethanol and a liquid acid catalyst to
generate a second reaction mixture at a biomass:catalyst ratio of
1:0.1 to 1:2 and a biomass:ethanol ratio of 1:1 to 1:10; and c.
heating the second reaction mixture to a temperature of
50-75.degree. C. for a period of 4-8 hours to generate an ester
mixture comprising at least some of the lipids converted into an
FAEE product.
2. The method of claim 1, further comprising cooling the ester
mixture to 30-50.degree. C.
3. The method of claim 2, further comprising neutralizing the ester
mixture with a weak base.
4. The method of claim 3, further comprising contacting the ester
mixture with a second non-polar solvent to generate a first
extraction mixture.
5. The method of claim 4, further comprising separating the first
extraction mixture into a first liquid fraction comprising the FAEE
product and a solid fraction comprising biomass; and recovering the
FAEE product in the first liquid fraction.
6. The method of claim 1, wherein the biological material comprises
algae.
7. The method of claim 6, wherein the algae is dried algae.
8. The method of claim 6, wherein the algae comprises at least one
species selected from the genera consisting of Schizochytrium and
Nannochloropsis.
9. The method of claim 1, wherein the first non-polar solvent
comprises at least one selected from the group consisting of
hydrocarbons, halogenated hydrocarbons, hexane, heptane, octane,
petroleum ether, chloroform, and supercritical carbon dioxide.
10. The method of claim 4, wherein the second non-polar solvent
comprises at least one selected from the group consisting of
hydrocarbons, halogenated hydrocarbons, hexane, heptane, octane,
petroleum ether, chloroform, and supercritical carbon dioxide.
11. The method of claim 4, wherein the first and second non-polar
solvent are the same.
12. The method of claim 4, wherein the first and second non-polar
solvent are different.
13. The method of claim 1, wherein the liquid acid catalyst
comprises at least one selected from the group consisting of
hydrochloric acid (HCl), boron trifluoride (BF.sub.3), phosphoric
acid (H.sub.3PO.sub.4), nitric acid, sulfuric acid, and organic
sulfonic acid.
14. The method of claim 3, wherein the weak base is water.
15. The method of claim 5, wherein the separation of the first
extraction mixture comprises at least one from the group consisting
of filtration, membrane filtration, and centrifugation.
16. The method of claim 5, further comprising fractionating the
FAEE product into a saturated FAEE product and an unsaturated FAEE
product.
17. The method of claim 16, wherein the fractionating is performed
by urea crystallization.
18. The method of claim 5, further comprising fractionating the
FAEE product into different length FAEE or different boiling point
FAEE fractions.
19. The method of claim 18, wherein the fractionating is performed
by molecular distillation.
20. The method of claim 18, wherein at least one fraction is
selected from the group consisting of an FAEE fraction having a
fatty acid carbon chain of 16 or less, an Omega-7 FAEE fraction, an
Omega-9 FAEE fraction, and an Omega-3 FAEE fraction.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of PCT Application No.
PCT/US2014/027161, filed Mar. 14, 2014, entitled Direct
Transesterification of Algal Biomass for Synthesis of Fatty Acid
Ethyl Esters (FAEE), U.S. Provisional Application No. 61/798,436,
filed Mar. 15, 2013, entitled Direct Transesterification of Algal
Biomass for Synthesis of Fatty Acid Ethyl Esters (FAEE), the entire
contents of which are hereby incorporated by reference.
BACKGROUND
[0002] Many high value products can be produced from algal oil
(e.g., microalgal oil, cyanobacteria oil), but obtaining the
products requires numerous processing steps, such as extraction and
transesterification, where efficiency and product mass can be lost
at each step in the process. For example, biodiesel production from
algal oil conventionally involves oil extraction followed by
transesterification to produce fatty acid methyl esters (FAME). The
majority of transesterification processes use a strong base to
catalyze the reaction because it only requires moderate conditions
and has a faster reaction time than an acid-catalyzed process,
which tends to be slower due to the equilibrium. An acid catalyzed
process is commonly used for biomass feedstocks with high free
fatty acid content where soaps are not desired, because these high
free fatty acid feedstocks may form soaps if a base catalyzed
process is utilized in an attempt to form esters. Enzymatic
transesterification is an emerging technology utilizing an enzyme
catalyst to produce FAME from algal oil, but currently is not
cost-effective due to issues with catalyst regeneration.
[0003] Direct transesterification (i.e., in-situ
transesterification) of algal biomass is less time consuming and is
more efficient than a conventional extraction transesterification
process due to the inherent nature of a single-stage reaction,
which comprises a reduction in process steps and material handling
where the target product may be lost. Direct transesterification
has been used (Johnson & Wen, 2009) to produce biodiesel from
Schizochytrium limacinum using an acid catalyzed
transesterification process with methanol, chloroform, hexane
and/or petroleum ether solvents. In-situ transesterification and
factors such as stirring, moisture content, and reaction
temperature were also studied for production of biodiesel in Ehimen
et. al (Ehimen, Sun, & Carrington, 2010). Biodiesel production
methods were simplified by Wagner et. al (Haas & Wagner, 2011)
and Mi et. al (Xu & Mi, 2010) using excess reagents and a
co-solvent strategy respectively. Currently direct
transesterification of algae technology focuses on production of
FAME for biodiesel using high temperature and excess solvents in an
inefficient manner.
[0004] Transesterification of triglycerides and fatty acids to
produce esters has been performed using catalyst/conditions, such
as: enzymes (Fjerbaek, Christensen, & Norddahl, 2009) (Modi,
Reddy, Rao, & Prasad, 2007) (Mata, Sousa, Vieira, &
Caetano, 2012); acid/base catalysts (Rodri & Tejedor, 2002)
(Alamu, Waheed, & Jekayinfa, 2008); or heterogeneous catalysts
(Zabeti, Wan Daud, & Aroua, 2009) (Liu, He, Wang, Zhu, &
Piao, 2008). Previously, specific fatty acids or their esters have
been purified from mixtures of fatty acids or their esters by
molecular distillation into a form that is more useful for end
products (Rossi, Pramparo, Gaich, Grosso, & Nepote, 2011)
(Tenllado, Reglero, & Tones, 2011).
[0005] The focus on direct esterification method development in the
biofuel art using methyl esters has not produced an efficient
method translatable to other high value products derived from algal
biomass. Therefore, there is a need in the art for an efficient
method of esterification for ethyl esters of high value products
from algal biomass.
SUMMARY
[0006] The instant invention describes methods of producing fatty
acid ethyl esters (FAEE) from lipid containing biological material.
The FAEE produced with the described methods may be used in a
variety of products including health, beauty, cosmetic, and
nutraceutical products.
[0007] In one embodiment of the invention, a method for converting
lipids in a lipid containing biological material into fatty acid
ethyl esters (FAEE) comprises: mixing biological material
comprising lipids and biomass with a first non-polar solvent with a
first non-polar solvent at a biomass:first non-polar solvent ratio
of 1:1 to 1:10 to form a first reaction mixture; mixing the first
reaction mixture with ethanol and a liquid acid catalyst to
generate a second reaction mixture at a biomass:catalyst ratio of
1:0.1 to 1:2 and a biomass:ethanol ratio of 1:1 to 1:10; and
heating the second reaction mixture to a temperature of
50-75.degree. C. for a period of 4-8 hours to generate an ester
mixture comprising at least some of the lipids converted into an
FAEE product. In some embodiments, the method further comprises:
cooling the ester mixture to 30-50.degree. C.; neutralizing the
ester mixture with a weak base; contacting the ester mixture with a
second non-polar solvent to generate a first extraction mixture;
separating the first extraction mixture into a first liquid
fraction comprising the FAEE product and a solid fraction
comprising biomass; and recovering the FAEE product in the first
liquid fraction.
[0008] In some embodiments, the biological material comprises
algae, and may further comprise dried algae. In some embodiments,
the first and second non-polar solvents may comprise at least one
selected from the group consisting of hydrocarbons, halogenated
hydrocarbons, hexane, heptane, octane, petroleum ether, chloroform,
and supercritical carbon dioxide. The first and second non-polar
solvents may be the same or different. In some embodiments, the
liquid acid catalyst may comprise at least one selected from the
group consisting of hydrochloric acid (HCl), boron trifluoride
(BF.sub.3), phosphoric acid (H.sub.3PO.sub.4), nitric acid,
sulfuric acid, and organic sulfonic acid. In some embodiments, the
weak base may be water. In some embodiments, the separation of the
first extraction mixture comprises at least one from the group
consisting of filtration, membrane filtration, and
centrifugation.
[0009] In some embodiments, the method may further comprise
fractionating the FAEE production into a saturated FAEE product and
an unsaturated FAEE product with a urea crystallization method. In
some embodiments, the method may further comprise fractionating the
FAEE production into different length FAEE or different boiling
point FAEE fractions with a molecular distillation method. The
different fraction may comprise an FAEE fraction having a fatty
acid carbon chain of 16 or less, an Omega-7 FAEE fraction , an
Omega-9 FAEE fraction, and an Omega-3 FAEE fraction.
[0010] In another embodiment of the invention, a method for
converting lipids in Schizochytrium biomass into fatty acid ethyl
esters (FAEE) comprises: mixing Schizochytrium biomass with a first
non-polar solvent, ethanol, and an acid catalyst to generate a
reaction mixture; heating the reaction mixture to generate an ester
mixture comprising at least some lipids converted into an FAEE
product; contacting the ester mixture with a second non-polar
solvent to generate a first extraction mixture; and separating the
first extraction comprising the FAEE product and a solid fraction
comprising biomass, wherein the FAEE product comprises an actual
FAEE yield of at least 89%. In some embodiments, the FAEE product
comprises an actual DHA FAEE yield of at least 85%.
[0011] In another embodiment of the invention, a method for
converting lipids in Nannochloropsis biomass into fatty acid ethyl
esters (FAEE) comprises: mixing Nannochloropsis biomass with a
first non-polar solvent, ethanol, and an acid catalyst to generate
a reaction mixture; heating the reaction mixture to generate an
ester mixture comprising at least some lipids converted into an
FAEE product; contacting the ester mixture with a second non-polar
solvent to generate a first extraction mixture; and separating the
first extraction comprising the FAEE product and a solid fraction
comprising biomass, wherein the FAEE product comprises an actual
FAEE yield of at least 69%. In some embodiments, the FAEE product
comprises an actual Omega-7 FAEE yield of at least 96%.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 shows side by side process flow diagrams for a
conventional extraction and transesterification process, and a
direct transesterification process.
[0013] FIG. 2 shows a diagram of the products resulting from a
process comprising direct transesterification and molecular
distillation of lipid containing biomass.
[0014] FIG. 3 shows a diagram of the products resulting from a
process comprising direct transesterification and urea
crystallization of lipid containing biomass.
[0015] FIG. 4 shows a diagram of the products resulting from a
sequential purification process comprising urea crystallization and
molecular distillation of polyunsaturated fatty acids (PUFA).
[0016] FIG. 5 shows a diagram of the products resulting from a
sequential purification process comprising molecular distillation
and urea crystallization of ester products.
DESCRIPTION OF THE INVENTION
[0017] The "lipid containing biological material" of the instant
invention may be any raw biological material or biomass from plant,
animal or microbial origin such as: oil seeds (e.g., rape, soybean,
sunflower, peanuts, walnuts, macadamia nuts, etc.); fruit (e.g.,
avocado, palm, coconut, etc.); plant tissues, such as stems, roots,
tubers and leaves (e.g., Jojoba, certain weeds, rotted food/feed,
algae, seaweed, kelp, etc.); animal tissues (e.g., adipose tissue,
organs, offal, slaughterhouse waste, insects, dead animals, etc.);
microorganisms and fermented products (e.g., microalgae, fungi,
bacteria, cyanobacteria, cheese, waste waters, fermented
agricultural products (silage, manure, wastes, etc.)); and mixtures
of any of these. The lipid containing biological material may be
fresh, stored, fermented, or decomposing. The lipid containing
biological material may be edible or inedible to humans or animals.
The animal, plant, and microorganism sources may be grown primarily
for this purpose of producing lipids, such as safflower seeds,
algae, microalgae, cyanobacteria, and insects; it may be a
byproduct of other processes (e.g., cold pressed soybean meal,
chicken feathers, etc.); or it may be wastes (e.g., sewage, food or
feed processing, etc.). The animal, plant, and microorganism
sources may be genetically modified organisms, specifically chosen
or designed for starch deficiency, high lipid content, an enhanced
fatty acid profile such as high C16 and C18 chain length fatty
acids content, and combinations thereof. The genetic modification
may be the result of selection, cell fusion, or transfer of one or
more genes from the same or different species.
[0018] The lipid containing biological material may be pure raw
material, mixed material, or containing other material with no
lipid such as an added adsorbent or carrier. Even lipid containing
biological material not typically thought of as having a high lipid
content, may still be used if so desired. The lipid containing
biological material may have been previously treated, such as to
remove lipids or other material provided that the bulk of the
biomass of the raw material remains. For example, microalgae
extracted with acetone to remove carotenoids or expeller pressed
soybeans to remove soybean oil, but the remaining microalgae
biomass or soybean meal may still contain at least some lipids.
This carotenoid depleted microalgae biomass or mostly delipidized
soybean meal may function as the lipid containing biological
material for the start of the process of the instant invention, but
the soybean oil would not. The lipid in the "lipid containing
biological material" may be in the form of free fatty acids or
attached by an ester bond to another chemical moiety such as a
mono, di or triglyceride or a wax.
[0019] The term "direct transesterification" refers to
transesterification of lipids in the lipid containing biological
material that contains considerable biomass. The term "direct
transesterification" has also been called "in-situ
transesterification" and the terms are usually used
interchangeably. "Indirect transesterification" or "conventional
extraction-transesterification" refers to transesterification of
previously extracted lipids such as oils, which contain relatively
little of the non-lipid biomass. Examples include solvent extracted
lipids, pressed lipids, rendered lipids, and synthetic lipids.
[0020] Conventional extraction-transesterification may be less
complex to perform because of the relatively homogenous nature of
the initial starting feedstock, but includes additional steps to
conduct the extraction separately from the transesterification
process. By comparison, direct transesterification contains
considerable biomass including high concentrations of complex
compounds of a very different chemical nature such as cellulose and
other carbohydrates, proteins, nucleic acids, salts, etc., but has
fewer steps. FIG. 1 shows a process flow for conventional
extraction-transesterification and direct transesterification side
by side, which highlights the reduction of process steps in the
direct transesterification process.
[0021] The term "algae" refers to phototrophic, mixotrophic, and
heterotrophic organisms such as green algae, cyanobacteria,
microalgae, unicellular algae, multicellular algae (e.g., duck
weed), diatoms, and dinoflagellattes.
[0022] The terms "phototrophic", "phototrophy", "photoautotrophy",
"photoautotrophic", and "autotroph" refer to culture conditions in
which light and inorganic carbon (e.g., carbon dioxide, carbonate,
bi-carbonate) may be applied to a culture of algae. Algae capable
of growing in phototrophic conditions may use light as an energy
source and inorganic carbon (e.g., carbon dioxide) as a carbon
source. An algae in phototrophic conditions may produce oxygen.
[0023] The terms "mixotrophic" and "mixotrophy" refer to culture
conditions in which light, organic carbon, and inorganic carbon
(e.g., carbon dioxide, carbonate, bi-carbonate) may be applied to a
culture of algae. Algae capable of growing in mixotrophic
conditions have the metabolic profile of both phototrophic and
heterotrophic organisms, and may use both light and organic carbon
as energy sources, as well as both inorganic carbon and organic
carbon as carbon sources. Mixotrophic algae may be using light,
inorganic carbon, and organic carbon through the phototrophic and
heterotrophic metabolisms simultaneously or may switch between the
utilization of each metabolism. Algae in mixotrophic culture
conditions may be a net oxygen or carbon dioxide producer depending
on the energy source and carbon source utilized by the algae. Algae
capable of mixotrophic growth comprise algae with the natural
metabolism and ability to grow in mixotrophic conditions, as well
as algae which obtain the metabolism and ability through
modification of cells by way of methods such as mutagenesis or
genetic engineering.
[0024] The terms "heterotrophic" and "heterotrophy" refer to
culture conditions in which organic carbon may be applied to a
culture of algae in the absence of light. Algae capable of growing
in heterotrophic conditions may use organic carbon as both an
energy source and as a carbon source. Algae in heterotrophic
conditions may produce carbon dioxide.
[0025] The invention comprises a method of direct
transesterification of algal biomass to produce a fatty acid ethyl
esters (FAEE) product, and methods of purifying the FAEE product.
The process utilizes a co-solvent method (reactant solvent and
non-polar solvent) reacting with dewatered or at least partially
dried algal biomass in the presence of a concentrated acid
catalyst. Algal biomass may be dewatered using centrifugation,
flocculation (e.g., polyelectrolyte or inorganic flocculants),
combined flocculation (i.e., using more than one type of
flocculant), autoflocculation, marine microalgal flocculation,
tangential flow filtration, gravity sedimentation, flotation (e.g.,
dissolved air flotation, dispersed air flotation), and
electrophoresis techniques (e.g., electrolytic coagulation,
electrolytic flotation and electrolytic flocculation). Dewatered
algae may be further dried by using one of the following
techniques: drum drying, spray-drying, sun-drying, solar-drying,
cross flow drying, vacuum shelf drying or freeze drying. While wet
algae may be used, the amount of water should be considered when
choosing the amount and concentration of acid catalyst and
ethanol.
[0026] Excess alcohol may be added and followed by evaporation of
the azeotrope to remove water. Alternatively, a water absorbent
polymer or composition, free water (A.sub.W) lowering compound, or
inert material may interact with and remove the water, such as a
salt that forms its hydrate in the presence of water. The reaction
is carried out at a relatively low reaction temperature. After the
reaction is completed, the reaction mixture is cooled, neutralized
with a base, and extracted with a non-polar solvent. The solid and
liquid fractions of the reaction mixture may be separated to
isolate the algal biomass in a solid fraction from the FAEE product
in a liquid fraction. The liquid fraction obtained after separation
may then be concentrated or purified to give a concentrated FAEE
product obtained from algal biomass suitable for use in health,
beauty, nutraceutical, and cosmetic products.
[0027] While the focus for algae derived products has primarily
been fuels, fatty acid methyl esters (FAME) for biodiesel is only
one high value product available from algae. Besides biodiesel,
algal can provide a feedstock for many other high value products
such as nutraceuticals, and cosmetics utilizing Omega 3, 6, 7 &
9 fatty acids. Some of these products may utilize ethyl esters, and
synthesis of ethyl esters is known as a method for enriching
Omega-3 fatty acids. Unlike the methyl esters of fatty acids, the
ethyl esters of fatty acids involve using less toxic compounds in
their synthesis and in the resulting product, making them
acceptable for human and animal uses.
[0028] Ethyl esters of lipid rich algal oil may be separated using
multiple separation techniques into multiple high value fractions,
such as: a) Fuel fraction (such as fatty acids of shorter carbon
chain length about C10 to C16); b) Omega-7 (such as C16:1n7,
C18:1n7, and C20:1n7 fatty acids) or Omega-9 fraction (such as
C18:1n9 and C22:1n9 fatty acids); c) Omega-3 fraction (such as
C20:5n3, C22:6n3, and C22:5n3 fatty acids); d) Microbial (including
algae) feed stock (such as very short carbon chain lengths of C10
or less); e) A combination of high value fractions, such as a
combination of an Omega-3 and Omega-7 fractions or the combination
of Omega-7 and Omega-9; and f) A fraction with a reduced content of
Omega-6 fatty acids (such as C18:2n6 and C20:4n6 fatty acids).
[0029] After separation, the fuel fraction may provide the input to
a hydrotreatment process for synthesis of a high cetane diesel
through hydrodeoxygenation treatments known in the art. The high
cetane diesel produced may subsequently be isomerized, using
methods known in the art, to give jet fuel. The Omega-7/9 fraction,
composed of Palmitoleic acid (C16:1n7) and Oleic acid (C18:1n9), is
a commercial product with many potential uses in the health,
medicine, and cosmetic industries. Omega-7 fatty acids (e.g.,
Palmitoleic acid) is found in human skin sebum and is known to
decline with age (Wille & Kydonieus, 2003). Omega-7 supplements
comprising sea buckthorn oil are currently available in the market
as a health product for skin and hair (contains approximately 30%
Omega 7) (Yang & Kallio, 2001). However, sea buckthorn oil is
limited in supply and alternative sources of Omega-7 are needed to
satisfy the growing demand for health, medicine, and cosmetic
products comprising Omega-7. Omega-7 ethyl esters derived from
algae, or non-algal sources such as macadamia nuts and menhaden,
may substitute for sea buckthorn oil in products, and provide an
advantage due to the fact that esters can be provided at a higher
purity not currently available from the oils in the market (Rusch
gen. Klaas & Meurer, 2004a). The Omega-7/9 fraction composition
is dependent on the algal species, with each species containing a
different fatty acid profile of varying quantities of C16:1, C18:1,
C18:2, C18:3, etc.
[0030] Omega-3 ethyl esters (e.g., Eicosapentaenoic acid (EPA) and
Docosahexaenoic acid (DHA)) are commercially important and are US
Food and Drug Administration (FDA) approved as antilipemic or
lipid-regulating agents. However, Omega-3 FAME is not currently FDA
approved. To satisfy the commercial demand, Omega-3 ethyl esters
are currently sourced from fish oil, which has a limited supply and
will continue to decline as the world consumption of fish
increases. Algal oil has potential as an attractive alternative to
the use of fish oil in Omega-3 products due to the availability of
algae and the lack of the odor associated with fish oils.
[0031] Non-limiting examples of algae that can be used with the
system and methods of the invention are members of one of the
following divisions: Chlorophyta, Cyanophyta (Cyanobacteria), and
Heterokontophyta. In certain embodiments, the algae used with the
methods of the invention are members of one of the following
classes: Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae.
In certain embodiments, the algae used with the methods of the
invention are members of one of the following genera:
Schizochytrium, Nannochloropsis, Chlorella, Dunaliella,
Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina,
Amphora, and Ochromonas.
[0032] Non-limiting examples of algae species that can be used with
the system and methods of the instant invention include: Achnanthes
orientalis, Agmenellum spp., Amphiprora hyaline, Amphora
coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis
var. punctata, Amphora coffeiformis var. taylori, Amphora
coffeiformis var. tennis, Amphora delicatissima, Amphora
delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus,
Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp.,
Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor,
Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis,
Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum,
Chaetoceros sp., Chlamydomas perigranulata, Chlorella anitrata,
Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida,
Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea,
Chlorella emersonii, Chlorella fusca, Chlorella fusca var.
vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella
infusionum var. actophila, Chlorella infusionum var. auxenophila,
Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis,
Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis
var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella
mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva,
Chlorella photophila, Chlorella pringsheimii, Chlorella
protothecoides, Chlorella protothecoides var. acidicola, Chlorella
regularis, Chlorella regularis var. minima, Chlorella regularis
var. umbricata, Chlorella reisiglii, Chlorella saccharophila,
Chlorella saccharophila var. ellipsoidea, Chlorella salina,
Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella
sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella
vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var.
autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris
var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia,
Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella,
Chlorella zofingiensis, Chlorella trebouxioides, Chlorella
vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium,
Chroomonas sp., Chrysosphaera sp., Cricosphaera sp.,
Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica,
Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella
bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella
maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei,
Dunaliella primolecta, Dunaliella salina, Dunaliella terricola,
Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta,
Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena
spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp.,
Galdieria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus
pluvialis, Hymenomonas sp., lsochrysis aff. galbana, lsochrysis
galbana, Lepocinclis, Micractinium, Micractinium, Monoraphidium
minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis
salina, Nannochloropsis sp., Navicula acceptata, Navicula
biskanterae, Navicula pseudotenelloides, Navicula pelliculosa,
Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis
sp., Nitschia communis, Nitzschia alexandrina, Nitzschia
closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia
frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia
intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia
pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia
quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva,
Oocystis pusilla, Oocystis sp., Oscillatoria limnetica,
Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri,
Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum,
Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae,
Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii,
Prototheca stagnora, Prototheca portoricensis, Prototheca
moriformis, Prototheca zopfii, Pseudochlorella aquatica,
Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid
chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra,
Spirulina platensis, Stichococcus sp., Synechococcus sp.,
Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron,
Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii,
and Viridiella fridericiana.
[0033] In other embodiments, the system and methods may use
non-algal oleaginous plant biomass. The non-algal oleaginous
biomass may be plant material, including but not limited to soy,
corn, palm, camelina, jatropha, canola, coconut, peanut, safflower,
cottonseed, linseed, sunflower, Jojoba, macadamia, hazelnut, rice
bran, and olive. Animal fats and synthetic fats and waste fats and
oils containing materials may also be used. In some embodiments,
the biomass may be at least partially dried to reduce the water
content of the biomass. In other embodiments, the biomass may be
completely dried (at least 90% solids).
[0034] The term "Omega 3" comprises polyunsaturated fatty acids of
carbon chain length C16:3n3 (hexadecatrienoic acid), C18:3n3
(.alpha.-Linolenic acid), C18:4n3 (Stearidonic acid), C20:3n3
(Eicosatrienoic acid), C20:4n3 (Eicosatetraenoic acid), C20:5n3
(Eicosapentaenoic acid (EPA)), C21:5n3 (Heneicosapentaenoic acid),
C22:5n3 (Docosapentaenoic acid/DPA), C22:6n3 (Docosahexaenoic acid
(DHA)), C24:5n3 (Tetracosapentaenoic acid), C24:6n3
(Tetracosahexaenoic acid), and the like.
[0035] The term "Omega 6" comprises unsaturated fatty acids of
carbon chain length C18:2n6 (Linoleic acid), C18:3n6
(Gamma-linolenic acid, Calendic acid), C20:2n6 (Eicosadienoic
acid), C20:3n6 (Dihomo-gamma-linolenic acid), C20:4n6 (Arachidonic
acid/AA), C22:2n6 (Docosadienoic acid), C22:4n6 (Adrenic acid),
C22:5n6 (Docosapentaenoic acid), C24:4n6 (Tetracosatetraenoic
acid), C24:5n6 (Tetracosapentaenoic acid), and the like.
[0036] The term "Omega 7" comprises unsaturated fatty acids of
carbon chain length C16:1n7 (Palmitoleic acid), C18:1n7 (Vaccenic
acid), C20:1n7 (Paullinic acid), and the like.
[0037] The term "Omega 9" comprises unsaturated fatty acids of
carbon chain length C18:1n9 (oleic acid, elaidic acid), C20:1n9
(gondoic acid), C20:3n9 (mead acid), C22:1n9 (erucic acid), C24:1n9
(nervonic acid), and the like.
Direct Transesterification Method
[0038] The synthesis of fatty acid ethyl esters (FAEE) is carried
out in the presence of ethanol as the reacting alcohol solvent.
Ethanol is in molar excess of the fatty acids being
transesterified. If water is present, ethanol should be in molar
excess of water. The biomass:ethanol ratio may be varied from about
1:1 to about 1:10. The co-solvent for the inventive process is a
non-polar solvent, generally hydrocarbons, such as halogenated
hydrocarbons, and may comprise some ethers such as hexane, heptane,
octane, petroleum ether, or chloroform. Supercritical carbon
dioxide may also be used as a co-solvent in a sealed reaction
vessel. It is desirable for the co-solvent to not be
unacceptability miscible in water or acid. It is also preferred
that the co-solvent not be unacceptably miscible in ethanol. The
biomass:non-polar solvent ratio may vary from about 1:1 to about
1:10. The invention minimizes the waste of solvents through the
optimization of the biomass:solvent ratios without losing
efficiency in the FAEE synthesis process.
[0039] The inventive process is acid catalyzed. The acid catalyst
may comprise hydrochloric acid (HCl), boron trifluoride (BF.sub.3),
phosphoric acid (H.sub.3PO.sub.4), nitric acid, sulfuric acid,
organic sulfonic acids, metal organic frameworks (e.g., zeolites
acting as Lewis or Bronsted acids), and other mineral, organic and
Lewis acids. Sulfuric acid is the most commonly used acid catalyst
for synthesis of fatty acid alkyl esters. The acid catalyst may be
in a gaseous or liquid form, and is preferably in a liquid form.
The ratio of biomass:acid catalyst may vary from about 1:0.1 to
about 1:2.
[0040] The reaction temperature for the inventive process is
approximately between 50-75.degree. C. depending on the type of
biomass, solvent type, and ratio selected for the reaction, and
preferable between about 60-65.degree. C. The inventive process
emphasizes the use of a lower reaction temperature when the
reaction is performed at atmospheric pressure. Higher temperatures
may be used in a pressurized reaction vessel. The goal is to
prevent the ethanol or its azeotrope from boiling or otherwise
being significantly volatilized. The reaction time may range from
about 4-8 hours, and is preferably about 6 hours.
[0041] The resulting product mixture of the reaction is cooled to
about 30-50.degree. C., and preferably to about 40.degree. C. The
cooled reaction mixture is neutralized with a weak base, such as
water, and extracted with a non-polar solvent, such as hexane. The
non-polar solvent used with the after the catalyzed ethanol
reaction may be the same or different from the co-solvent used
earlier in the process. After a non-polar solvent extraction, the
solid and liquid fractions are separated using known methods such
as filtration, membrane filtration, or centrifugation to separate
the solid fraction comprising the biomass from the liquid fraction
comprising the crude FAEE product. The crude FAEE product comprises
a mixture of saturated and unsaturated fatty acids of a proportion
which is dependent on the profile of the biomass feedstock
used.
[0042] In one embodiment of the invention, a method of converting
lipids contained in an algal biomass into fatty acid ethyl esters
(FAEE) using co-solvents and isolating a high purity FAEE product
comprises: drying algal biomass containing lipids; mixing the dried
algal biomass with a first non-polar solvent; contacting the dried
algal biomass and first non-polar solvent with a solvent comprising
ethanol and a catalyst comprising an acid to generate a first
reaction mixture at a biomass:acid catalyst ratio less than about
1:0.6; heating the first reaction mixture to a temperature less
than about 90.degree. C. for a sufficient period of time to convert
at least some of the lipids into an FAEE product through direct
transesterification to generate an ester mixture; cooling the ester
mixture; neutralizing the ester mixture with a weak base;
contacting the ester mixture with a second non-polar solvent to
generate a first extraction mixture; separating the first
extraction mixture into a first solid fraction and a first liquid
fraction comprising the FAEE product; and purifying the FAEE
product in the first liquid fraction.
[0043] After separation of the non-polar phase containing FAEE, the
polar phase containing at least the unreacted ethanol may be
further treated to remove and recover ethanol. This may be done by
volatilizing or boiling ethanol or the azeotrope from the polar
phase by using heat and/or vacuum. The ethanol may be recovered and
optionally recycled into the initial direct transesterification
process.
[0044] After separation, the non-polar solvent may be recovered by
boiling or volatilizing it from the FAEE. This non-polar solvent
may be recycled in the process. Other materials of value may also
be recovered such as the glycerol, acid, or biomass residue after
the direct transesterification process.
[0045] Without being bound by a single theory, results of the
direct transesterification method may be dictated by the nature of
the oil in the starting biological material. For example, direct
transesterification may be affected by the lipid profile present in
algal biomass; and based on the observations of the experimental
runs direct transesterification may most efficient when the neutral
lipid content (e.g., triglycerides) in the algal biomass is high.
Algae species are also known to have polar lipids (e.g.,
phospholipids, glycolipids), for these species the use of hexane
(i.e., hydrophobic solvent) in the process may be substituted with
hydrophilic solvents, such as but not limited to, chloroform,
carbon tetrachloride, etc.
Separation and Purification Methods
[0046] After formation of the crude FAEE product, further
separation and purification may produce higher purity products for
fuel, health, beauty, cosmetic, and nutraceutical products.
[0047] In the context of the instant invention, simple distillation
is the crude separation of FAEE from most other lipids, solvents,
unreacted oils in the feedstock and other unwanted materials that
are in the FAEE containing fraction. No fractionation to
distinguish between different FAEEs is intended other than perhaps
extremely short or extremely long FAEE outside the desirable ranges
mentioned above.
[0048] The method of the instant invention may also use a
separation technique to separate the fatty acid esters based on
chain length, size, and the like. Molecular distillation is a
convenient example of a separation and purification technique that
may be used for separation of FAEE in the process of the instant
invention. Molecular distillation includes a process with a short
exposure of a distilled liquid to elevated temperatures, at least
partial vacuum in the distillation space, and a short path between
the condenser and evaporator. Molecular distillation is a process
commonly used to purify oils and is a suitable process for use in
conjunction with algae derived ester products of the instant
invention. Molecular distillation is also known to provide
advantages for natural products where the toxicity of other solvent
based separation methods may compromise the product, and may
operate at a lower pressure than vacuum distillation. The molecular
distillation technique may also be used to separate FAEE and other
fatty acid esters into high value fractions, such as a fuel
fraction, Omega-7/9 fraction, and an Omega-3 fraction as shown in
FIG. 2.
[0049] In the instant invention, the fractionation of fatty acid
ester process may include a separation of saturated fatty acids
from unsaturated fatty acids. While chromatography and other
techniques may be used, urea crystallization is particularly good
for such a separation. Urea crystallization (Wanasundara &
Shahidi, 1999) (Shahidi & Wanasundara, 1998) (Belarbi, 2001) is
another technique which may be utilized in the instant invention to
separate and purify FAEE fractions. Urea inclusion compound (UIC)
based fractionation of free fatty acids may be applied to algae
derived FAEE. Urea complexes form between urea molecules and
"guest" molecules, typically saturated fatty acids for algae oil
based applications. For the instant invention, the FAEE are the
"guest" molecules and they function similarly to fatty acids. Urea
crystallization provides a simple and efficient technique to
separate saturated fat from more valuable Omega (3, 6, 7 or 9)
fatty acids as shown in FIG. 3.
[0050] In a urea crystallization separation, concentration, and/or
purification process for FAEE synthesized by direct
transesterification, first the crude FAEE product is treated with
urea in the presence of ethanol. The resulting reaction mixture is
refluxed at about 80.degree. C. for about 2 hours. After the
reflux, the reaction mixture is cooled to room temperature
(approximately 18-24.degree. C.) and then kept in a refrigerator at
about 4.degree. C. overnight (a period of about 7-12 hours). The
refrigeration step is used to ensure complete crystallization,
which comprises the formation of urea crystals suspended in a
polyunsaturated fatty acid (PUFA) rich liquid phase. The
crystallized suspension is then filtered, preferably under vacuum,
to separate the urea crystals and the PUFA rich filtrate. The PUFA
rich filtrate may be washed with water to remove urea, and is
extracted with a non-polar solvent such as hexane. The non-polar
solvent layer is then separated, such as by a centrifuge, and is
concentrated, such as under vacuum on a rotary evaporator, to give
a PUFA rich FAEE product comprising Omega 3, 6, 7 and/or 9 fatty
acids.
[0051] The separated urea crystals are dissolved in hot water (at
about 90.degree. C.) for 2 hours. The reaction mixture is then
cooled to about 40.degree. C. and is extracted using a non-polar
solvent, such as hexane. The non-polar solvent layer is separated
using a centrifuge and is concentrated on a rotary evaporator
resulting in a saturated fat rich FAEE product comprising C14, C16,
and C18 fatty acids.
[0052] Urea crystallization may be performed using methanol or
ethanol as an alcohol solvent. The ratio of biomass:urea may vary
from about 1:1 to 1:10 while the biomass:ethanol ratio may vary
from about 1:2 to 1:50. The crystallization temperature of the urea
crystallization process depends on the solvent used and may vary
between about 60-85.degree. C.
[0053] If the separation of saturated FAEE from unsaturated FAEE is
insufficient for the desired product(s), the process may be
repeated any number of times with either the urea filtrate fraction
or the urea crystals fraction to obtain a better separation.
[0054] The separation and purification methods described above may
be used individually or in combination to obtain a desired fatty
acid containing fraction. The fatty acid containing fraction may be
FAEE, FAME, C3-6 alcohol esters of fatty acids or even mixtures of
these. In one embodiment, the unsaturated fatty acids or their
esters obtained by urea crystallization may be further purified by
molecular distillation to obtain valuable fatty acid containing
fractions such as Omega-7 and Omega-3 (i.e. Eicosapentaenoic acid,
Docosahexaenoic acid) as shown in FIG. 4. A simple distillation
prior to the urea crystallization may be used to provide cleaner
separations in the urea crystallization and/or the molecular
distillation.
[0055] Molecular distillation may refine the products into two,
three, or more different fractions as mentioned above or molecular
distillation may be used in combination with urea crystallization
to further purify the fatty acid fractions obtained from the
molecular distillation process as shown in FIG. 5. A simple
distillation may be used initially to clean up the FAEE containing
composition to allow better separation by molecular distillation
and urea crystallization. Example 5 and Table 5 show the results
from molecular distillation of Nannochloropsis derived FAEE. The
Omega-7 and Omega-3 fractions shown in Table 5 may be further
purified using urea crystallization by removing saturated fatty
acid containing compounds, which may be detrimental for various
dietary, cosmetic, nutraceutical, and pharmaceutical applications.
This method of using molecular distillation followed by urea
crystallization may also be applied to mixtures of FAME, FAEE, C3-5
alcohol esters of fatty acids and mixtures of these.
[0056] The direct transesterification method may function in
substantially the same manner in a large scale production context.
The molar ratio of chemicals, temperature, and reaction temperature
are all applicable to reactions of different volumes.
Alternate Transesterification Protocols
[0057] While the following examples involve acid catalysts for
transesterification, another embodiment of the instant invention is
the partial transesterification by an acid catalyst followed by
further transesterification by alkali. This is particularly
preferable when the lipid containing biological material contains
significant amounts of free fatty acids, such as greater than about
5% free fatty acids, more preferably more than about 10% free fatty
acids. Alkali catalyzed transesterification is generally faster and
involves less specialized equipment than acid catalysis, but forms
soaps in the presence of free fatty acids. In experiments with an
algae feedstock containing free fatty acids and using a direct
sulfuric acid catalyzed transesterification followed by direct
sodium hydroxide alkali catalyzed transesterification, FAEE was
produced in high yields without formation of soaps.
[0058] The use of acid catalyzed transesterification of at least
the free fatty acids followed by alkali catalyzed
transesterification of the remaining fatty acids is believed to be
applicable to feedstocks by indirect transesterification as well as
direct transesterification. It is also believed that other lower
alcohols may be used in either the direct or the indirect
transesterification using the combined acid catalyzed following by
alkali catalyzed transesterification process.
EXAMPLE 1
Direct Transesterification of Schizochytrium limacinum
[0059] Dried algal biomass of the species Schizochytrium limacinum
(400 g) was mixed with hexane (800 mL, resulting in a
biomass:non-polar solvent ratio of 1:2) in a 5 L round bottom
flask. An ethanol-sulfuric acid solution was prepared separately by
mixing concentrated sulfuric acid (128 mL, resulting in a
biomass:acid catalyst ratio of 1:0.32) with ethanol (800 mL,
resulting in a biomass:ethanol ratio of 1:2) with constant
stifling. The ethanol-sulfuric acid solution was mixed with the
biomass-hexane solution with constant stirring in a 5 L round
bottom flask to generate a reaction mixture. The reaction mixture
was then refluxed at 60.degree. C. for 6 hours for completion of
reaction. Completion of a reaction may be determined by thin layer
chromatography (TLC). After 6 hours, the reaction mixture was
cooled to 40.degree. C. and was neutralized by water (800 mL). The
reaction mixture was then extracted using hexane (800 mL), and the
extraction was performed three times to ensure complete extraction.
The algal biomass, hexane (product) layer and aqueous layer were
separated using a centrifuge at 25.degree. C. with 6,000 rpm for 5
minutes. The hexane layer was then concentrated using a rotary
evaporator to give the crude FAEE product. The yield and recovery
were calculated as listed below:
% Yield = Product Yield Starting amount .times. 100 ##EQU00001##
Actual recovery = % Yield .times. % Purity 100 ##EQU00001.2## %
Actual Yield = Actual Recovery Oil Content .times. 100
##EQU00001.3## starting amount = amount of algae ##EQU00001.4##
product yield = amount of oil ##EQU00001.5## actual recovery =
purities of oil in total biomass ##EQU00001.6##
[0060] The direct transesterification procedure described in
EXAMPLE 1 for the 400 g biomass sample was performed in the same
manner with the same solvent types, catalyst types, solvent ratios,
catalyst ratios, temperatures, and reaction times for all of the
experimental runs using 25 g, 40 g, and 400 g of biomass as listed
in TABLE 1. The results in TABLE 1 show yield of 61.75-67.36%,
purity of 74.02-86.53%, DHA of 16.91-19.5%, actual yield of at
least 89.29%, and DHA (actual) 83.65-97.96%.
TABLE-US-00001 TABLE 1 Starting % % Amount % Actual DHA (g) Yield %
Purity % DHA Yield (actual) 25 67.04 Reaction 80.41 18.98 98.73
97.22 25 63.4 time 6 hrs 76.9 17.6 89.29 85.26 25 67.36 74.02 16.91
91.32 87.03 25 66.48 81.3 18.82 98.99 95.6 40 65.025 78.5 17.92
93.49 89.03 40 65.75 86.53 19.5 104.2 97.96 400 66.75 84.89 19.12
103.78 97.52 400 61.75 79.99 17.73 90.46 83.65
EXAMPLE 2
Urea Crystallization For Separation of FAEE Based on
Unsaturation
[0061] The crude FAEE product (40 g) synthesized by direct
transesterification of Schizochytrium limacinum was mixed with urea
(80 g, resulting in a product:urea ratio of 1:2) and ethanol (200
mL, resulting in a product:ethanol solvent ratio of 1:5) in a 500
mL round-bottom flask. The reaction mixture was refluxed at
80.degree. C. for 2 hours. After 2 hours the reaction mixture was
cooled to room temperature (18-24.degree. C.) and was then kept in
a refrigerator (approximately 4.degree. C.) overnight
(approximately 8 hours) to ensure complete crystallization. The
crystallized mixture was then filtered under vacuum to separate
urea crystals and a PUFA rich filtrate. The urea crystals were also
washed with cold ethanol (200 mL, a product:ethanol ratio of 1:5)
to ensure complete separation. The PUFA rich filtrate was washed
with water (200 mL, a product:water ratio of 1:5) and was extracted
using hexane (200 mL, a product:hexane ratio of 1:5) to separate a
PUFA rich FAEE product from soluble urea. The hexane layer was
separated using a centrifuge at 25.degree. C. with 6,000 rpm for 5
minutes and was concentrated on a rotary evaporator to give a PUFA
rich FAEE product with a high concentration of C22:6
docosahexaenoic acid (DHA).
[0062] The urea crystals were dissolved in water (200 mL, resulting
in a product:water ratio of 1:5) at 90.degree. C. for 2 hours.
After 2 hours, the reaction mixture was cooled to 40.degree. C. and
was extracted using hexane (200 mL, a product:hexane ratio of 1:5).
The hexane layer was separated using a centrifuge at 25.degree. C.
with 6,000 rpm for 5 minutes and was concentrated on a rotary
evaporator to give a saturated fat rich FAEE product enriched with
C14, C16 and C18 fatty acids. The experiment as described was run
three times. TABLE 2 displays the results for the process described
in EXAMPLE 2. The results in TABLE 2 show a urea filtrate recovery
of 37.6-45.0%, urea crystal recovery of 47.5-61.7%, a total
recovery of at least 89.6%, a urea filtrate with 67.9-73.9% of
polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids
(MUFA), a urea filtrate with 2.23-6.58% saturated fatty acids
(SFA), urea crystals with 6.29-13.00% of polyunsaturated fatty
acids (PUFA) and monounsaturated fatty acids (MUFA), and urea
crystals with 63.64-82.79% saturated fatty acids (SFA).
TABLE-US-00002 TABLE 2 Urea Urea Urea Filtrate Urea crystals
Filtrate Crystals % Total % PUFA + % PUFA + % Recovery % Recovery
Recovery MUFA % SFA MUFA % SFA 43.7 61.7 100.0 67.90 5.19 8.40
63.64 37.6 52.0 89.6 73.90 2.23 13.00 72.97 45.0 47.5 92.5 69.50
6.58 6.29 82.79
EXAMPLE 3
Direct Transesterification of Nannochloropsis
[0063] Dried algal biomass of the species Nannochloropsis sp. (40
g) was mixed with hexane (80 mL, resulting in a biomass:non-polar
solvent ratio of 1:2) in a 500 mL round bottom flask. An
ethanol-sulfuric acid solution was prepared separately by mixing
concentrated sulfuric acid (128 mL, resulting in a biomass:acid
catalyst ratio of 1:0.32) with ethanol (80 mL, resulting in a
biomass:ethanol ratio of 1:2) with constant stifling. The
ethanol-sulfuric acid solution was mixed with the biomass-hexane
solution with constant stirring in a 5 L round bottom flask to
generate a reaction mixture. The reaction mixture was then refluxed
at 63.degree. C. for 6 hours for completion of reaction. Completion
of a reaction may be determined by thin layer chromatography (TLC).
After 6 hours, the reaction mixture was cooled to 40.degree. C. and
was neutralized by water (80 mL). The reaction mixture was then
extracted using hexane (80 mL), and the extraction was performed
three times to ensure complete extraction. The algal biomass,
hexane (product) layer and aqueous layer were separated using a
centrifuge at 25.degree. C. with 6,000 rpm for 5 minutes. The
hexane layer was then concentrated using a rotary evaporator to
give the crude FAEE product. The yield and recovery were calculated
as listed below:
% Yield = Product Yield Starting amount .times. 100 ##EQU00002##
Actual recovery = % Yield .times. % Purity 100 ##EQU00002.2## %
Actual Yield = Actual Recovery Oil Content .times. 100
##EQU00002.3##
[0064] The direct transesterification procedure described in
EXAMPLE 3 for the 40 g biomass sample was performed in the same
manner with the same solvent types, catalyst types, solvent ratios,
catalyst ratios, temperatures, and reaction times for all of the
experimental runs using 40 g of Nannochloropsis biomass in oil
production phase, and 50 g of Nannochloropsis biomass in growth
phase as listed in TABLE 3. Results of the yield, purity, Omega-7,
and Omega-3 percentages are listed in TABLE 3.
TABLE-US-00003 TABLE 3 Starting Amount (g) 40 50 Species (stage)
Nannochloropsis Nannochloropsis (oil) (Growth) Temp .degree. C. 63
63 % Yield 60 14 % Purity 54.4 65.88 % .omega.-7 17.99 16 %
.omega.-3 2.54 18.19 % Actual Yield 69.2 46.3 % .omega.-7 (actual)
96.0 79.9 % .omega.-3 (actual) 63.7 105.4
EXAMPLE 4
Urea Crystallization For Separation of Fatty Acid Ethyl Esters
(FAEE's) Based on Unsaturation
[0065] The crude FAEE product (34 g) synthesized by direct
transesterification of Nannochloropsis sp. was mixed with urea (68
g, resulting in a product:urea ratio of 1:2) and ethanol (340 mL,
resulting in a product:ethanol solvent ratio of 1:10) in a 500 mL
round-bottom flask. The reaction mixture was refluxed at 80.degree.
C. for 2 hours. After 2 hours the reaction mixture was cooled to
room temperature (18-24.degree. C.) and was then kept in a
refrigerator (approximately 4.degree. C.) overnight (approximately
8 hours) to ensure complete crystallization. The crystallized
mixture was then filtered under vacuum to separate urea crystals
and PUFA rich filtrate. The urea crystals were also washed with
cold ethanol (200 mL, a product:ethanol ratio of 1:5) to ensure
complete separation. The PUFA rich filtrate was washed with water
(200 mL, a product:water ratio of 1:5) and was extracted using
hexane (170 mL, a product:hexane ratio of 1:5) to separate PUFA
rich FAEE product from soluble urea. The hexane layer was separated
using a centrifuge at 25.degree. C. with 6000 rpm for 5 minutes and
was concentrated on a rotary evaporator to give a PUFA rich FAEE
product enriched in Omega-3 and Omega-7 fatty acids.
[0066] The urea crystals were dissolved in water (170 mL, resulting
in a product:water ratio of 1:5) at 90.degree. C. for 2 hours.
After 2 hours, the reaction mixture was cooled to 40.degree. C. and
was extracted using hexane (170 mL, a product:hexane ratio of 1:5).
The hexane layer was separated using centrifuge at 25.degree. C.
with 6000 rpm for 5 minutes and was concentrated on a rotary
evaporator to give a saturated fat rich FAEE product enriched in
C14, C16, and C18 fatty acids. The results for the experiment as
described in EXAMPLE 4 are displayed in TABLE 4.
TABLE-US-00004 TABLE 4 Urea Urea Urea Filtrate Urea crystals
Filtrate Crystals % % % % % Total PUFA + PUFA + Recovery Recovery
Recovery MUFA % SFA MUFA % SFA 42.6 41.8 84.4 61.56 14.52 14.62
44
EXAMPLE 5
[0067] A molecular distillation method for separation of fatty acid
ethyl esters derived from Nannochloropsis sp. (as described in
Example 3) was performed. The results listed below in TABLE 5
demonstrate that molecular distillation is a suitable method for
separating the Omega-7 and Omega-3 fractions of an algae derived
FAEE product.
TABLE-US-00005 TABLE 5 Molecular Distillation Omega-7 Omega-3
Fraction fraction Total Ethyl Esters (%) 98.53 93.9 Total Saturates
(%) 49.79 11.17 Total Monounsaturates (%) 41.73 28.72 Total
Polyunsaturates (%) 2.1 49.36 Total Omega-3 (%) 0.66 33.37 Total
Omega-6 (%) 1.44 Total Omega-9 (%) 5.93 22.78 Total Omega-7 (%)
35.13 4.79
Comparative Examples
[0068] The following comparative examples compare known methods of
direct transesterification with the instant invention.
EXAMPLE 6
[0069] Johnson et al. (Johnson & Wen, 2009) investigated direct
transesterification using a co-solvent system. The process focused
on synthesis of biodiesel (i.e., FAME) and needs further
optimization for use in producing high purity fatty acid ethyl
esters (FAEE). The high reaction temperature (90.degree. C.) used
in the Johnson et al. process is often used to check feasibility of
the process, but causes unnecessary solvent loss and violent reflux
during the reaction. Therefore, the high temperature process
disclosed by Johnson et al. lacks an efficient use of catalyst
(i.e. sulfuric acid), hexane, and reactant solvent (i.e., methanol)
resources. The process disclosed by Johnson et al. also does not
address the production of high purity FAEE for use in health and
beauty products that would require FDA approval, which is lacking
for an FAME based product.
[0070] TABLE 6 below compares the process parameters for the
process disclosed by Johnson et al. to the instant invention, and
highlights the distinctions in the resultant product, amount of
catalyst (i.e., sulfuric acid) required, amount of hexane required,
type of reactant solvent, amount of reactant solvent, reaction
temperature, and reaction time. From TABLE 6, the more efficient
use of energy, catalyst, and solvent resources of the co-solvent
system of the instant invention is apparent, as well as the
production of a product suitable to be purified and used in health
and beauty applications.
TABLE-US-00006 TABLE 6 Johnson and Instant Wen 2009 Invention
Product FAME FAEE Catalyst Sulfuric acid Sulfuric acid
Biomass:Catalyst ratio 1:0.6 1:0.32 Biomass:Hexane ratio 1:4 1:2
Reactant Solvent Methanol Ethanol Biomass:Reactant 1:4 1:2 Solvent
ratio Reaction 90.degree. C. 60.degree. C. Temperature Reaction
Time 1.5 hrs 6 hrs Algal biomass tested 400 g 10-400 g
EXAMPLE 7
[0071] Haas et al. (Haas & Wagner, 2011) investigated the
comprehensive process optimization for direct transesterification
of algal biomass. The process proposed by Haas et al. focused on
synthesis of an FAME biodiesel product, not FAEE, and is also
performed on an analytical scale not easily translatable to
commercial production. The Haas et al. process is performed using a
single solvent system, which when compared to the process disclosed
by Johnson et al. above has shown that a single-reacting solvent
system is less efficient than a two solvent system in achieving
product yield. The Haas et al. process uses excess reacting solvent
(methanol) and sulfuric acid to achieve high yields, which is
inefficient and costly. Therefore, Haas et al. also does not
provide an efficient process for achieving high yields of FAEE for
use in health and beauty products.
[0072] TABLE 7 below compares the process parameters for the single
solvent process disclosed by Hass et al. to the co-solvent process
of the instant invention, and highlights the distinctions in the
resultant product, amount of catalyst (i.e., sulfuric acid)
required, type of solvents, amount of reactant solvent, and
reaction time. From TABLE 7, the more efficient use of catalyst and
solvent resources of the instant invention is apparent, as well as
the production of a product suitable to be purified and used in
health and beauty applications at a scale more suitable to
commercialization.
TABLE-US-00007 TABLE 7 Hass and Instant Wagner 2011 Invention
Product FAME FAEE Catalyst Sulfuric acid Sulfuric acid 15-32 mmoles
= 1.47-3.14 g (calculated from molecular weight 98.078 g/mol)
Biomass:Catalyst ratio 1:0.59-1:1.26 1:0.32 Biomass:Hexane ratio
N/A 1:2 Reactant Solvent Methanol 8-20 ml Ethanol Biomass:Reactant
1:3.2-1:8 1:2 Solvent ratio Reaction Temperature 23-65.degree. C.
60.degree. C. Reaction Time 2 hrs 6 hrs Algal biomass tested 2.5 g
10-400 g
EXAMPLE 8
[0073] Ehimen et al. (Ehimen et al., 2010) investigated physical
parameters such as stifling, temperature, and reaction time for the
production of biodiesel products (i.e., FAME). Ehimen et al.
emphasized the importance of moisture content and the negative
impact of moisture on the yield, and also illustrated a direct
transesterification process utilizing Chlorella. The process
disclosed by Ehimen et al. utilizes less sulfuric acid, but still
uses a high amount of alcohol reactant solvent because the process
is a less efficient single solvent system (as opposed to a
co-solvent system). Additionally, the process disclosed by Ehimen
et al. demonstrated better yields at a high reaction temperature of
90.degree. C. and pressure of 3 bar, which requires more energy
than process conducted at a lower temperature and atmospheric
pressure. The higher reaction temperature and pressure allow for
the possibility for instability to be introduced into the reaction
when reacting a larger quantity of algal biomass. Therefore, Ehimen
et al. also does not provide an efficient process for achieving
high yields of FAEE for use in health and beauty products.
[0074] TABLE 8 below compares the process parameters for the single
solvent process disclosed by Ehimen et al. to the co-solvent
process of the instant invention, and highlights the distinctions
in the resultant product, type of solvents, amount of reactant
solvent, and reaction time. From TABLE 8, the more efficient use of
solvent resources of the instant invention is apparent, as well as
the production of a product suitable to be purified and used in
health and beauty applications at a scale more suitable to
commercialization.
TABLE-US-00008 TABLE 8 Instant Ehimen 2010 Invention Product FAME
FAEE Catalyst Sulfuric acid 2.2 ml Sulfuric acid Biomass:Catalyst
ratio 1:0.15 1:0.32 Biomass:Hexane ratio NA 1:2 Reactant Solvent
Methanol 20-100 ml Ethanol Biomass:Reactant 1:4 1:2 Solvent ratio
Reaction Temperature 23-90.degree. C. 60.degree. C. Reaction Time
0.25-12 hrs 6 hrs Algal biomass tested 15 g 10-400 g
EXAMPLE 9
[0075] Harvey et al. (Velasquez-Orta, Lee, & Harvey, 2012)
disclosed an alkaline in-situ transesterification process for
Chlorella vulgaris. The process disclosed by Harvey et al. used a
reduced amount of alkaline catalyst and had a shorter reaction time
than in an acid catalyst system, but used a significantly larger
amount of reactant solvent (i.e., methanol) than an acid catalyst
system. The in-situ alkaline transesterification also focused on
production of biodiesel products (i.e., FAME), and was shown to
result in lower yields than an acid catalyst system. Harvey et al.
also used a single solvent system, which has shown to be less
efficient than a co-solvent system. Therefore, Harvey et al. also
do not provide an efficient process for achieving high yields of
FAEE for use in health and beauty products.
[0076] TABLE 9 below compares the process parameters for the single
solvent, alkaline catalyst process disclosed by Harvey et al. to
the co-solvent, acid catalyzed process of the instant invention,
and highlights the distinctions in the resultant product, type of
catalyst, type of solvents, amount of reactant solvent, and
reaction time. From TABLE 9, the more efficient use of solvent
resources of the instant invention is apparent, as well as the
production of a product suitable to be purified and used in health
and beauty applications at a scale more suitable to
commercialization.
TABLE-US-00009 TABLE 9 Instant Harvey et al. 2012 Invention Product
FAME FAEE Catalyst Sodium hydroxide Sulfuric acid Biomass:Catalyst
ratio 1:0.15 1:0.32 Biomass:Hexane ratio NA 1:2 Reactant Solvent
Methanol Ethanol Biomass:Reactant 1:600 1:2 Solvent ratio Reaction
Temperature 60.degree. C. 60.degree. C. Reaction Time 1.25 hrs 6
hrs Algal biomass tested 7 g 10-400 g
EXAMPLE 10
[0077] US 2012/0065416 A1 describes a method for converting
microbial lipids from an oleaginous microbial biomass into fatty
acid alcohol esters, without prior extraction of the lipids from
the biomass (in situ transesterification) to produce FAME. The
examples in the reference primarily use methanol, but ethanol is
evaluated in example 8 of US 2012/0065416 A1. In the described
method of US 2012/0065416 A1, examples 8 and 9 show the efficiency
of the process can depend on the ability of alcohol to extract the
lipids. To ensure adequate lipid extraction, excess reactant
solvents (i.e., alcohols) and sulfuric acid are used, which is an
inefficient use of resources. US 2012/0065416 A1 also uses a single
solvent system which has shown to be less efficient than a
co-solvent system. Therefore, US 2012/0065416 A1 also does not
provide an efficient process for achieving high yields of FAEE for
use in health and beauty products.
[0078] TABLE 10 below compares the process parameters for the
single solvent process disclosed by US 2012/0065416 A1 to the
co-solvent process of the instant invention, and highlights the
distinctions in the resultant product, type of solvents, amount of
reactant solvent, and reaction time. From TABLE 10, the more
efficient use of solvent resources of the instant invention is
apparent, as well as the production of a product suitable to be
purified and used in health and beauty applications.
TABLE-US-00010 TABLE 10 Instant US 2012/0065416 A1 Invention
Product FAME FAEE Catalyst Sulfuric acid Sulfuric acid
Biomass:Catalyst ratio 1:0.05-1:0.12 1:0.32 Biomass:Hexane ratio NA
1:2 Reactant Solvent Methanol Ethanol Biomass:Reactant 1:1-1:100
1:2 Solvent ratio Reaction Temperature 60-110.degree. C. 60.degree.
C. Reaction Time 1-10 hrs 6 hrs Algal biomass tested 100 g 10-400
g
[0079] As shown in the above discussion, the prior art methods do
not provide a commercially scalable high yield method for producing
an ester product which conserves energy, catalyst, and solvent
resources. Therefore there is a need in the art for an efficient
commercial scale process for converting oil derived from algae into
fatty acid ethyl esters (FAEE) and purifying the FAEE for use in
health and beauty products.
[0080] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific embodiments described specifically
herein. Such equivalents are intended to be encompassed in the
scope of the following claims.
* * * * *