WO2011100563A2

WO2011100563A2 - Transesterified lipid recovery and methods thereof

Info

Publication number: WO2011100563A2
Application number: PCT/US2011/024549
Authority: WO
Inventors: Sridhar Viamajala; Daniel Nelson; Ronald Sims
Original assignee: Utah State University
Priority date: 2010-02-12
Filing date: 2011-02-11
Publication date: 2011-08-18
Also published as: WO2011100563A3

Abstract

Methods for the direct extraction and transesterification of cellular lipids in biological material are described. The transesterified lipids produced during this process form a distinct phase from the rest of the reactants and products, and this phase can be easily separated to recover the lipid fractions without the need for further solvent extraction or distillation. The method produces transesterified lipids suitable for use as biodiesel and nutritional products, including polyunsaturated fatty acids such as omega-3 or -6 fatty acids. Additionally, the transesterified lipid products may be further processed to obtain other high purity lipids, without the need for further solvent extraction or distillation.

Description

TRANSESTERIFIED LIPID RECOVERY AND METHODS THEREOF Technical Field

[0001] The present disclosure relates to the recovery of lipids from algal and other similar biological material, and their conversion to products such as biodiesel or other fatty acid alkyl esters. More specifically, a reactive extraction is performed on the biomass to effectively recover transesterified cellular lipids as a separate phase.

Background

[0002] In recent years, the significance of lipids has increased due to their applications in renewable energy as well as for nutrition and health. Lipids from sources such as vegetable oil can be converted to biodiesel through a transesterification reaction that produces fatty acid alkyl esters. Most often, fatty acid methyl esters (FAMEs) are used as biodiesel due to the relatively lower cost of methanol, one of the reactants in the process. With increasing demand for biodiesel, conventional vegetable oil feedstocks derived primarily from oilseeds such as soy, canola, palm, rapeseed, and sunflower, are unable to meet market needs, resulting in an unsustainable pressure on agricultural practices and food sources. As a result, alternative feedstocks for lipids are needed. In addition to plants, microorganisms, such as algae or fungi, are also capable of accumulating lipids (including mono-, di- and triglycerides of fatty acids). In fact, laboratory research shows that algae are more efficient lipid producers than terrestrial plants and can yield up to two orders of magnitude higher lipid mass per acre per year than most conventional oil crops.

[0003] Algae and other microorganisms are also capable of synthesizing large amounts of polyunsaturated fatty acids (PUFAs) that are important for mammalian health and nutrition. PUFAs in the body help to maintain proper organ function. For example, high docosahexanoic acid (DHA) content in the brain and retina has been shown to be important for proper nervous system and visual function. PUFAs are likely essential ingredients in an infant diet and are added into most commercially available baby formula foods. PUFAs such as omega-3 fatty acids can also act as breast and colon cancer chemopreventive agents.

[0004] The technologies that exist for extracting oils from plants and converting them to fuel and/or nutritive products may not necessarily apply to microbial biomass because of the differences in their physical properties. Therefore, new production technologies which are efficient and cost effective on a large scale, must be developed for microbial systems.

Brief Description of the Drawings

[0005] Figure 1 is a photomicrograph of Schizochytrium limacinum SR21 cells used in accordance with embodiments of the present invention.

[0006] Figure 2 is a gas chromatograph showing the lipid profile of Schizochytrium limacinum SR21 cells after solvent extraction in accordance with embodiments of the present invention. The peaks in the 30-35 min region represent triacylglycerides (TAGs). The peak at 20.5 min corresponds to the octacosane internal standard.

[0007] Figure 3 is a gas chromatograph showing the fatty acid methyl ester (FAME) profile Schizochytrium limacinum SR21 cells after transesterification of cellular lipids in accordance with embodiments of the present invention. The TAG peaks in the 30-35 min region are absent confirming their conversion to FAMEs. The peak at 20.5 min corresponds to the octacosane internal standard. The peaks corresponding to the major FAMEs (C14:0. C15:0, C16:0 and DHA) are labeled.

[0008] Figure 4 is the time course of total FAME yield during in-situ transesterification at different temperatures in accordance with embodiments of the present invention.

[0009] Figure 5 is the time course of total FAME yield during in-situ transesterification at different acid concentrations in accordance with embodiments of the present invention.

[0010] Figure 6 is the time course of total FAME yield during in-situ transesterification at different biomass concentrations in accordance with embodiments of the present invention.

[0011] Figure 7 is a photograph of duplicate reaction vials after the in-situ transesterification reaction at 90°C in 5% acidified methanol solution containing 250 mg/mL biomass, in accordance with embodiments of the present invention. The floating lipid layer is clearly visible.

[0012] Figure 8 is a gas chromatograph of the lipid layer of Figure 7, showing that fatty acid methyl esters (FAMEs) are the dominant chemical species in this phase. [0013] Figure 9 is a graph which shows the FAME content of the methanol phase and the entire reactor contents, in accordance with embodiments of the present invention.

[0014] Figure 10 is a graph which shows the FAME recovery with recycled solvent at different biomass concentrations after 3h at 90°C in 5% acidified methanol solution, in accordance with embodiments of the present invention.

[0015] Figure 1 1 is a photograph of duplicate reaction vials after the in-situ transesterification reaction using recycled solvent, in accordance with embodiments of the present invention. The floating lipid layer is clearly visible.

[0016] Figure 12 is a graph comparing the fatty acid alkyl ester yield from an in- situ transesterification in ethanol and methanol under similar reaction conditions, in accordance with embodiments of the present invention.

[0017] Figure 13 shows the conventional mechanism of transesterification of TAGs in the presence of an acid catalyst.

[0018] Figure 14 is a graph comparing the experimental data and model predictions for the transesterification reactions carried out at various biomass concentrations, in accordance with embodiments of the present invention.

[0019] Figure 15 is a graph of the variation of the reaction kinetic parameter, V_M, with acid concentration, in accordance with embodiments of the present invention.

Detailed Description

[0020] Conventional analytical methods for the extraction of cellular lipids commonly use the well-known Bligh and Dyer method. In this procedure, a mixture of solvents, such as chloroform, methanol, and water is used to perform the extraction and the solvent ratios are balanced such that a single phase is formed with the aqueous sample. After completion of the extraction, phase separation is achieved by diluting the mixture with chloroform and water, allowing the extracted lipids contained in the chloroform phase to be removed. Known modifications or adaptations of this procedure involve changing the solvents and their ratios. For example, methylene chloride has been used as an alternative to chloroform and variations in extraction time, temperature, and order of solvent addition may be beneficial. Another emerging practice is the use of sonication during the extraction. Sonication helps to disrupt the cells and facilitates faster lipid release from cells, but this method adds significant process costs. Microwaves can also be used to expedite the extraction process, also with additional costs. Within all the adaptations of the Bligh and Dyer method, however, the principle mechanisms of extraction remain the same.

[0021] There are several disadvantages of the Bligh and Dyer solvent extraction followed by the transesterification method described above. For example, toxic and/or chlorinated solvents are used in this process, which will need to be effectively recovered and recycled. Solvent recovery is an expensive process and adds to the cost of biodiesel production. Also, even with recovery processes in place, there is a high likelihood of generating hazardous waste that will incur additional treatment costs. In addition, the process requires two steps: an extraction step to obtain lipids from biomass, followed by a reaction step to convert the lipids to biodiesel. Additional steps might be needed as well, to recover the biodiesel from the solvent to provide a "fuel-ready" product. Further, due to the use of toxic and/or chlorinated solvents, the process might be unsuitable for production of human nutrition products (including PUFAs), and the multiple steps involved lead to long processing times. Overall, the known solvent extraction based methods require multiple energy- intensive steps, incur high disposal costs and are unsuitable for generation of nutritive products.

[0022] Supercritical fluid extraction (SFE) using carbon dioxide is an alternative approach that significantly lowers the requirements for organic solvents, and mitigates concerns for waste disposal after the reaction. However, conditions affecting SFE, such as particle size and water content, must be improved to achieve efficient extractions. Also, SFE requires a high initial capital investment and continuously high energy inputs, which are two factors that render this method prohibitively expensive, especially for commodity products such as biofuels.

[0023] Lab scale transesterification reactions have been reported to provide an analytical description of cellular lipids (also known as a lipid profile). In these tests, a low biomass to reactant (acidified methanol) ratio is used, on the order of approximately 20 mg biomass per ml_ of reaction. The FAMEs generated in such methods are low in concentration and are completely soluble in the reaction mixture. If a scale-up based on these existing protocols were to be done for the commercial production of transesterified lipids, a separate solvent extraction (e.g., using hexane or chlorinated solvents) would first be needed to retrieve the FAMEs from the reaction mixture. The resulting FAME/solvent solution would then have to undergo additional processing, such as distillation, to recover the solvent. In this step using hexane as the solvent, the hexane would boil off at ~70°C, if carried out under atmospheric pressure, leaving behind an un-evaporated FAME product. Thus, this method would still require multiple steps (reaction, extraction and distillation) and the use of an additional solvent. Also, if this process were to yield a human nutritive product to generate an additional revenue stream, the distillation bottoms containing FAMEs would have to be very carefully stripped of all residual solvent, which would require yet additional processing. Finally, processing biomass at the low concentrations described in conventional protocols would require prohibitively large- scale equipment. As an example, if algal biomass containing 50% lipid (by weight) were to be reacted at 20 mg/mL, a reaction vessel with a 100 gallon volume would be needed to produce one gallon of product.

[0024] The transesterification methods disclosed herein mitigates the need for solvent and reduces the overall processing time by combining the extraction and transesterification into a single step. In these methods, biomass is treated with a mixture of alcohol and acid such that the reactive extraction releases and converts cellular lipids to fatty acid alkyl esters including, but not limited to, FAMEs.

[0025] Base-catalyzed reactions have been more extensively studied because they are faster than acid-catalyzed reactions and the reaction conditions are moderate. However, use of alkali for the transesterification of lipids from whole cell biomass can result in formation of soaps from cellular free fatty acids (FFAs). The soaps lower the product quality and necessitate further downstream purification. In addition, the presence of water (such as from incompletely dried biomass) during base-catalyzed reactions causes the hydrolysis of alkyl esters into FFAs, which also adds to the formation of soap. If acid catalysis is used, soap formation can be avoided since the FFAs are also converted to alkyl esters. Although acid-catalyzed transesterification generally requires higher temperatures and longer reaction times than base-catalyzed methods, the overall cost savings due to higher product quality make acid-catalyzed transesterification useful for FAME production from microbial biomass.

[0026] In the methods disclosed herein, the acid-catalyzed in-situ transesterification reaction conditions have been optimized such that the FAMEs form a separate liquid phase from the rest of the reaction mixture and can be easily separated. Without limiting the invention in any way, methods of separation include, but are not necessarily limited to, simple operations such as "skimming" or pumping out the individual phases.

[0027] The formation of at least two phases may occur through settling under gravity and the spontaneous coalescence of lipid droplets, or it may be facilitated by the application of an external force. An external force is additional energy exerted to separate the phases, and includes centrifugation. The formation of at least two phases may also be facilitated by an external chemical agent, which may be added before, during, or after the reaction. The external chemical agent may be water, an inorganic salt, a polar organic compound, a non-polar organic compound, and an alcohol-soluble organic or inorganic acid.

[0028] The lipids may be further refined after separation, for example, by distillation, to produce a fuel-grade biodiesel product. Alternatively, a nutritive lipid product may be isolated from the lipids, such as, for example, an omega fatty acid, a carotenoid, a sterol, or a wax.

[0029] The acid catalyst and the unreacted alcohol may be recycled to carry out the reaction multiple times, without the excess use of chemicals and with minimal disposal costs. The methods disclosed herein can process high biomass concentrations (up to nearly-saturated slurries) that may result in considerable cost savings through the reduction in equipment size and the high throughput.

[0030] The unreacted biomass, likely rich in protein and other non-lipid nutrients, may also be recovered separately from the transesterified lipids as a co-product to facilitate an additional revenue stream. For example, the co-product may be a protein-rich animal feed, a feedstock for aerobic or anaerobic fermentation, a food additive, or a bio-based material or composite.

EXAMPLES

[0031] The invention will be further described with references to the following examples. The examples are intended only to be illustrative, but not to limit the scope of the invention in any sense.

[0032] EXAMPLE 1 .

[0033] In an embodiment, an acid-catalyzed in-situ transesterification reaction method is disclosed, which results in the recovery of cellular triacylglycerols (TAGs) as FAMEs. Greater than 80% of the FAMEs form a separate liquid phase from the rest of the reaction mixture that can be easily separated through simple operations such as "skimming" or pumping out of the individual phases. The methods disclosed herein eliminate the need for subsequent solvent extractions and distillation. The methods involve reacting lipid-containing biomass with an acidified methanol solution.

[0034] Optimal conditions for conversion and phase separation were determined by a systematic study of the variables affecting the in-situ transesterification reaction. These parameters include time, temperature, acid concentration and biomass concentration.

[0035] Dried Schizochytrium limacinum SR21 (ATCC MYA 1381 ) cells were used as a model lipid-containing biomass source. A photomicrograph of these cells is shown in Figure 1 . The cells were grown in standard 5L spinner flasks containing 4L of growth media. The growth media contained 1 g/L yeast extract, 1 g/L peptone, 10g/L sea salt and 6g/L glycerol. The medium was autoclaved, adjusted to a pH of 7.0 and inoculated using 100 ml_ of previously grown liquid culture. The spinner flasks were incubated at room temperature on stir-plates to provide mixing. Sterile air was sparged through the cultures to provide oxygen for heterotrophic growth on glycerol. After 5 days of incubation, late log-phase cultures were harvested by centrifuging the reactor contents. The cell paste was washed twice with a 0.85% NaCI solution to remove residual media components. Washed biomass was then lyophilized and stored at -80°C.

[0036] In an embodiment, the biomass source may be a microalgae, a macroalgae, a yeast, a fungus, an oil seed, an oil seed processing residue, other plant matter including low-cellulosic matter, or an animal tissue such as meat, lard or waste trimmings. Combinations or blends of biomass sources may also be used, such as a mixed algal culture.

[0037] The cellular fatty acid content and composition was determined using standard analytical methods. Gas chromatography with flame ionization detection (GC-FID) was used to measure the concentration of fatty acids and mass spectroscopy (GC-MS) was used to identify the transesterified lipids. These data provided baseline information that could be used to determine the extent of conversion and FAME production during later experiments. Chromatograms corresponding to the lipid analysis of SR21 are shown in Figures 2 and 3. Figure 2 shows a chromatogram of a "total lipid extract" performed with a mixture of chloroform, tetrahydrofuran and hexane (in a volumetric ratio of 1 :1 :1 ) and shows that the lipids were mostly TAGs that eluted from the column between 30-35 minutes. Figure 3 is a chromatogram corresponding to samples where the lipids were converted to FAMEs by conventional in-situ transesterification techniques using acidified methanol. The absence of peaks in the TAG region of the chromatogram (retention time of 30-35 min) confirmed that complete conversion of TAGs to FAMEs was achieved during this analysis. GC-MS analysis of the peaks in Figure 3 showed that the major fatty acids were palmitic acid (C 16:0) and docosahexanoic acid (DHA, C22:6) while small amounts of myristic (C14:0) and pentadecanoic (C15:0) fatty acids were also present. Based on calibrations performed with high purity analytical standards, the exact cellular concentration of each of these lipids was determined and is shown in Table 1 .

[0038] Table 1 : FAME components in SR21 cells relative to total dry cell (biomass) weight.

14:0 15:0 16:0 22:6

(Myristic) (Pentadecanoic) (Palmitic) (DHA)

(mg/mg-biomass) (mg/mg-biomass) (mg/mg-biomass) (mg/mg-biomass)

0.009±0.005 0.006±0.007 0.243±0.009 0.237±0.006

[0039] From these data, the total lipid content of SR21 in the cultures was determined to be nearly 50% on a dry cell weight basis (g-lipid/g-biomass).

[0040] Studies on the effect of temperature were done in the range of 60°C to 100°C. These tests were carried out using biomass concentrations of 66 mg/mL. Mixtures of biomass and acidified methanol (containing 5% v/v H2SO4) were heated to target temperatures and the concentration of FAMEs produced was measured over time. FAME measurements were done using GC-FID after extraction of the entire reactor contents with known volumes of hexane. The results of this study are shown in Figure 4. These data show that the rates of reaction increase with increasing temperature. At 60°C, complete conversion to FAMEs was not achieved within the duration of this experiment. At 100°C, additional peaks were observed in the chromatograms, suggesting the formation of possibly undesirable by-products. Consequently, it was concluded that temperatures near 90°C are appropriate for this reaction. [0041] In an embodiment, a temperature between about 60°C and about 100°C may be used for the transesterification reaction. In an additional embodiment, a temperature between about 85°C and about 95°C may be used. In a further embodiment, a temperature of about 90°C may be used.

[0042] Different acid concentrations were tested. These were varied between 1 and 10% v/v and the reactions were carried out at 90°C with 66 mg/mL of biomass. Again, mixtures of biomass and acidified methanol were incubated over time and the FAME production was measured. The effects of acid concentration on the reaction rate and extent can be seen in Figure 5. Conversion of TAGs to FAMEs was observed in all experiments. However, the rates of reaction increased with increasing acid concentration and tests carried out in 5% acidified methanol solution resulted in the fastest reaction. In the presence of 10% acid (not shown), the reaction results were similar to those obtained at 5%. However, reactions using a 5% acid concentration were chosen as an optimum acid level.

[0043] In an embodiment, an acid concentration between about 1 % and about 10% may be used for the transesterification reaction. In an additional embodiment, an acid concentration between about 2% and about 5% may be used. In a further embodiment, an acid concentration of about 5% may be used. In still a further embodiment, an acid concentration of at least 1 % may be used.

[0044] The acid used includes standard organic and inorganic acids, for example, sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, or a combination or mixture thereof. In an embodiment, the acid is sulfuric acid.

[0045] The effects of biomass concentration on the reaction were studied. These tests were carried out at 90°C using an acidified methanol solution with 5% sulfuric acid (v/v). Results shown in Figure 6 demonstrate that conversion was achieved in nearly the same amount of time with 125 or 200 mg/mL biomass. Experiments with 250 mg/mL biomass reached completion over a slightly longer period.

[0046] Experiments performed in 5% acidified methanol solutions with biomass concentrations of 100 mg/mL or higher showed lipid droplets forming a separate "light phase" from the rest of the reaction mixture. However, good phase separation of lipids was observed to occur with biomass concentrations of 200 mg/mL or higher, as shown in Figure 7. These conditions also correspond to the highest biomass concentrations tested. Beyond these concentrations, the biomass slurry starts to become thick and paste-like. More viscous slurries may be difficult to mix and are likely to have higher associated energy costs.

[0047] In an embodiment, a biomass concentration between about 60 mg/mL and about 600 mg/mL may be used for the transesterification reaction. In an additional embodiment, a biomass concentration between about 66 mg/mL and about 250 mg/mL may be used. In a further embodiment, a biomass concentration between about 125 mg/mL and about 200 mg/mL may be used. In still a further embodiment, a biomass concentration of at least 125 mg/mL may be used.

[0048] GC-FID analysis of the lipid phase using 5% acid for time intervals 10, 20 and 30 minutes is shown in Figure 8 and confirms that FAMEs were the major components. The magnified inset illustrates the disappearance of TAGs over time, as the peak areas decrease as the time increases.

[0049] Analysis of the total and methanol-soluble FAMEs showed that the phase separation of lipids likely occurred due to the limited solubility of the FAMEs in the post-reaction acidified methanol solution. These results are shown in Figure 9. The concentration of FAMEs in the acidified methanol phase stayed constant at approximately 23 mg/mL for all the tests, while the overall FAME production was higher in the reaction vessels containing higher biomass concentrations. When FAME concentrations exceed the solubility limit in acidified methanol, spontaneous phase separation of the lipids occurs. The phase separation may be facilitated by the addition of an external force, such as centrifugation of the reactor contents.

[0050] Since the limited solubility of FAMEs in the acidified methanol solution is the most likely cause for phase separation of the transesterified lipids, it is possible in other embodiments to use external agents to decrease the solubility of lipids in the acidified alcohol solution and improve the phase separation behavior. Such additives may include water, common salts (e.g. sodium chloride, calcium sulfate, etc.), organic compounds (esters, aldehydes, ketones) or soluble acids. These could be added before, during, or after the reaction. Mixtures of or blends of the additives may also be used.

[0051] Overall, the methods disclosed herein are low cost methods of producing FAMEs from cellular lipids. The phase separation of a substantial portion of the lipids in reactions carried out at high biomass concentrations facilitates the easy recovery of FAMEs without the use of additional solvents. In addition, the "high solids processing" disclosed herein improves the overall process economics by increasing throughput and lowering process equipment volumes.

[0052] EXAMPLE 2.

[0053] In an embodiment, the acid catalyst and the unreacted alcohol may be recycled to carry out the reaction multiple times. This may prevent the excess use of chemicals and minimize disposal costs.

[0054] To carry out the in-situ transesterification reactions with recycled acidified methanol, the spent reactants (including the remaining methanol, acid catalyst, soluble FAMEs and other by-products of the reaction) were carefully recovered from the reaction vials of Example 1 after completion of the first transesterification reaction. This recycled reactant product was first filtered to remove any residual cell debris, then used to carry out a second set of in-situ transesterification reactions. The second set of reactions using recycled acidified methanol were carried out at biomass concentrations of 66, 125, 200 and 250 mg/mL. The lipid yield was determined after appropriately accounting for soluble lipids from the previous reactive-extraction step. The results of FAME recovery with recycled reactant are shown in Figure 10. The data show that nearly 100% recovery of lipids was obtained when low biomass concentrations were used. At high biomass concentrations, at least 85% of the lipids were transformed into FAMEs during the 3h reaction period. This would likely be improved if longer reaction times were used or if fresh acidified methanol was supplemented to the reaction mixture. A photograph of duplicate vials with the lipid layer on the surface of the acidified methanol phase is shown in Figure 1 1 .

[0055] EXAMPLE 3.

[0056] The recovery of FAMEs in the lipid layer has been determined to be independent of the type of fatty acid. This implies that low concentrations of high- value nutritive lipids are quantitatively recoverable from the lipid phase. If a preferential partition of certain lipids were to occur into the methanol phase, products including, but not limited to omega fatty acids, carotenoids, sterols, waxes and other hydrophobic components might be more difficult to recover.

[0057] The in-situ transesterification reactions were carried out in 5% acidified methanol solution using biomass concentrations of 66, 125, 200 and 250 mg/mL. The composition of the total FAMEs in the reaction vessel as well as in the acidified methanol phase were determined by GC-FID. The FAME composition of the lipid phase was calculated to be the difference between the overall concentration of individual FAMEs in the reaction vial (the total FAME amount, or TFA) and the methanol-soluble FAMEs. Results of the analysis are shown in Table 2. Based on this data, it is evident that the composition of both the soluble and insoluble phases is nearly the same and there is no preferential solubilization (or separation) of any single fatty acid.

[0058] Table 2: Composition of the different FAMEs in the methanol (soluble) and lipid (insoluble) phases as well as the total FAMEs produced. The results show that no favorable partitioning of FAMEs occurs in either phase.

Biomass DHA C16 C14 C15 Total

Fatty

Acids

(mg mL-1 ) (mg mL-1 ) (mg mL-1 ) (mg mL-1 ) (mg mL-1 ) (mg mL-1 )

66 Soluble 9.61 1 1 .1 1 0.30 0.16 21 .17

ln-sol. 6.59 5.63 0.31 0.16 12.70

TFA 16.20 16.74 0.61 0.32 33.87

125 Soluble 10.76 1 1 .80 0.25 0.12 22.93

ln-sol. 17.31 16.56 1 .48 0.82 36.17

TFA 28.08 28.36 1 .73 0.94 59.1 1

200 Soluble 10.91 12.27 0.42 0.21 23.81

ln-sol. 41 .07 34.75 2.86 1 .33 80.02

TFA 51 .99 47.02 3.28 1 .55 103.83

250 Soluble 1 1 .61 10.56 0.56 0.28 23.01

ln-sol. 50.72 45.27 3.08 1 .88 100.96

TFA 62.34 55.83 3.64 2.16 123.97 [0059] EXAMPLE 4.

[0060] In an embodiment, the residual biomass, likely rich in protein and other non-lipid nutrients, is recovered separately from the transesterified lipids. This may facilitate an additional revenue stream from the process. The solid biomass residue after the end of the reaction settles to the bottom of the vessel. As the FAME layer can be skimmed off from the top, the residual biomass can be recovered from the bottom of the reaction vessel or a subsequent settling tank.

[0061] EXAMPLE 5.

[0062] In an embodiment, other alkyl esters of fatty acids may be obtained by performing in-situ transesterification reaction with other alcohols. These alcohols include, but are not limited to, ethanol, propanol, isopropyl alcohol and butanol. Mixtures or blends of the alcohols may also be used.

[0063] Figure 12 shows the time course of formation of fatty acid alkyl esters along with a comparison of the rate of formation of FAMEs. The rates of formation of ethyl esters are almost identical to those of FAME formation.

[0064] EXAMPLE 6.

[0065] In an embodiment, the in-situ transesterification of TAGs to FAMEs may be described using a mathematical model derived from first principles based on known reaction mechanisms. Such a model is scale-independent and can be used for the future design of large-scale reactors.

[0066] The actual acid catalyzed reaction mechanism is shown in Figure 13. This can be re-written as a series of reactions as:

S + H+ SH+

S5¾F - MethylEster * Glycerol where S= TAG, S' = methanol, and the k's denote the various rates of the forward and reverse reactions. [0067] The following rate expressions can then be written for the elementary reactions in the overall mechanism

k S] [H⁺] = k^S ^]

k₂ [SH⁺] [S'] = k₂'[SS'H⁺]

<t[S\

dt

Where the variables stand for:

[S] = specific stable TAG concentration at any time during the reaction

(mg of TAG per ml_ of methanol);

[H⁺] = specific stable H⁺ concentration at any time during the reaction

(mg of H⁺ per ml_ of methanol);

[S'] = is assumed to be a constant throughout the reaction;

[SH⁺] = specific stable TAG-H⁺ complex at any time during the reaction

(mg of TAG-H⁺ per ml_ of methanol);

[SS'H⁺] = specific stable TAG-H⁺-MeOH complex at any time during the reaction (mg of TAG-H⁺-MeOH per ml_ of methanol); and

dP/dt = the rate of product production.

[0068] Since the first two reactions are reversible, the equilibrium constants may be written as

. [S][H⁺]

[0069] From these relationships,

dP d[S]

dt dt k_akb [S] [H⁺] [S']

[0070] Since [H⁺] is unknown during the reaction, a mass balance of total concentration in the reactor can be written as:

[H⁺]₀ = [H⁺] + [SH⁺] + [SS'H⁺]

[0071] Substituting and eliminating unknown variables, then

[0072] Also, the constant terms can be reassigned to single parameters through the following additional substitutions,

k-a

Sr ^, m

k b

[0073] Under the experimental conditions, methanol concentrations [S'] were much higher than the concentration of intracellular TAGs [S] and can be assumed to be constant. Also, if experiments are performed using a fixed initial acid concentration (i.e. [H⁺]₀ is 5%), then K_M and V_M can be treated as constants. In terms of these new constants, the rate equation for the formation of FAMEs is il ill ψ

[0074] From this expression, the rate of formation of FAMEs is dependent only upon the concentration of intracellular TAGs. The mathematical model was fit to experimental data where FAME production was measured over time as a function of biomass concentration, using acidified methanol containing 5% v/v H 2SO4. Single values of VM and KM that resulted in the best fit were determined by minimizing the sum of the square of errors between experimental and predicted values. The results of the mathematical model fit are shown in Figure 14. It can be seen that a close correlation with experimental results was obtained using the model. Best-fit values of VM and KM were determined to be 1 .43 mg per ml_ per min and 23.15 mg/mL, respectively.

[0075] To further verify its validity, the mathematical model was tested on experiments that were performed at constant biomass concentrations but had varying initial acid concentrations. Since KM is independent of initial acid concentration, it was fixed at the previously obtained value of 23.15 mg/mL. VM was determined independently for each of the experiments carried out at 1 , 1 .5, 2 and 5% acid concentration and 66 mg/mL biomass. As before, this was done by fitting the overall rate expression to the experimental data by minimizing the sum of square of differences between the measured and predicted values. From the expression of VM, it can be seen that the value of this parameter is directly proportional to initial acid concentrations, if the model was valid. Values of VM obtained from best-fit model predictions were plotted against initial acid concentrations used in the experiment and these results are shown in Figure 15. The strong correlation obtained again validates the applicability of the mathematical model.

[0076] As the rate expression was derived from first principles, the mathematical model development and validation studies show that FAME production can be accurately described using fundamental reaction kinetics that are scale-independent. Thus, a priori design of large-scale reactors is possible by extrapolation of the laboratory data for in-situ transesterification of microbial biomass.

[0077] EXAMPLE 7.

[0078] In an embodiment, a method for the in-situ transesterification of intracellular lipids for the production of biodiesel was tested on a mixed culture of algae obtained from the Logan, Utah municipal waste water lagoons. The mixed algal culture used was grown in biofilm form, harvested, and lyophilized prior to being transesterified to FAMEs. A total lipid extraction of the algae was also performed in order to determine the total lipid content of the algae.

[0079] The in-situ transesterification was performed according the procedures disclosed herein. Conditions and parameters used for the reaction included the following: a biomass to solvent ratio of 50 mg:1 mL; a sulfuric acid concentration of 5% v/v in methanol; and a reaction temperature of 90°C for 60 min. The reaction was performed in triplicate and FAME yields from the biomass were measured using gas chromatography. Data from the samples analyzed are shown below in Table 3, as the resulting FAME yields from the mixed culture algae present in the waste water lagoons.

[0080] Table 3. FAME yields from mixed culture algae present in the Logan, UT Lagoons

[0081] The yield of FAMEs from the mixed culture algae in the municipal waste water lagoons of Logan, UT, show that mixed algal cultures are viable as a source of biomaterial for the production of fatty acid methyl esters.

[0082] EXAMPLE 8.

[0083] In an embodiment, a total lipid extraction analysis was performed on the samples of Example 7 to compare the FAME results produced by the in-situ transesterification methods described herein, with total lipid yield. This information was used to determine the percent of total lipids that were converted to FAMEs, corresponding to biodiesel, using the disclosed methods. The analysis used for total lipid determination was a variation of the Folch total lipid extraction method. Results of the total lipid analysis are presented in Table 4. [0084] Table 4. Results of the total lipid extractions from the mixed culture algae.

[0085] The yield of total lipids from the mixed culture algae in the municipal waste water lagoons of Logan, UT, show that FAME yields are a significant portion of the total lipid yields, and that mixed algal cultures are viable as a source of biomaterial for the production of fatty acid methyl esters.

[0086] Although the invention has been illustrated by way of the above Examples thereof, it will be appreciated by those skilled in the art that various changes, alterations and modifications may be made to the invention without departing from the spirit and scope of the present disclosure as claimed.

Claims

1 . A method of obtaining transeste fied lipids from biological material comprising the steps of:

reacting the biological material with an alcohol and an acid; and

incubating the mixture,

wherein the reacting and incubating steps result in the formation of at least two phases, wherein the first phase comprises transestehfied lipids and the second phase comprises an acidified alcohol solution.

2. The method of claim 1 , wherein the reacting and incubating steps further result in the formation of a third phase, which comprises biomass residue.

3. The method of claim 1 , wherein the formation of at least two phases occurs through settling under gravity and the spontaneous coalescence of lipid droplets.

4. The method of claim 1 , wherein the formation of at least two phases is facilitated by centrifugation.

5. The method of claim 1 , wherein the formation of at least two phases is facilitated by an external chemical agent, wherein the external chemical agent is selected from at least one of the following: water, an inorganic salt, a polar organic compound, a non-polar organic compound, and an alcohol-soluble organic or inorganic acid.

6. The method of claim 1 , wherein the biological material is selected from at least one of the following: a microalgae, a macroalgae, a mixed algal culture, a yeast, a fungus, an oil seed, an oil seed processing residue, other plant matter, and an animal tissue.

7. The method of claim 1 , wherein the biological material comprises Schizochytrium limacinum SR21 (ATCC MYA 1381 ).

8. The method of claim 1 , wherein the concentration of the biological material is between about 66 mg/mL and about 250 mg/mL.

9. The method of claim 1 , wherein the alcohol is selected from at least one of the following: methanol, ethanol, propanol, isopropyl alcohol, butanol, and mixtures thereof.

10. The method of claim 1 , wherein the acid is selected from at least one of the following: sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, and mixtures thereof.

1 1 . The method of claim 1 , wherein the acidified alcohol solution of the second phase is further recovered and re-used for at least one subsequent application of the method of claim 1 .

12. The method of claim 1 , further comprising the step of refining the transesterified lipids to produce a fuel-grade biodiesel product.

13. The method of claim 1 , further comprising the step of isolating a product selected from at least one of the following: an omega fatty acid, a carotenoid, a sterol, and a wax.

14. The method of claim 2, further comprising the step of recovering from the solid phase a co-product, wherein the co-product is selected from at least one of the following: a protein-rich animal feed, a feedstock for aerobic or anaerobic fermentation, a food additive, and a bio-based material or composite.

15. The method of claim 1 , wherein the reacting or incubating step occurs at an elevated temperature.

16. The method of claim 15, wherein the elevated temperature is between about 60°C and about 100°C.

17. A method of obtaining fatty acid alkyl esters from biological material comprising the steps of:

reacting the biological material with an alcohol and an acid; and

incubating the mixture,

wherein the reacting and incubating steps result in the formation of at least two phases, wherein the first phase comprises fatty acid alkyl esters and the second phase comprises an acidified alcohol solution.

18. The method of claim 17, further comprising the step of isolating the fatty acid alkyl esters without the use of a chlorinated solvent.

19. The method of claim 17, wherein the alcohol is methanol.

20. A method of obtaining fatty acid methyl esters from biological material comprising the steps of:

reacting the biological material with an alcohol and an acid; and

incubating the mixture,

wherein the reacting and incubating steps result in the formation of at least two phases, wherein the first phase comprises fatty acid methyl esters and the second phase comprises an acidified alcohol solution.