# Patent

## Title
Systems and methods for extracting lipids from wet algal biomass

## Abstract

Presented herein are exemplary systems and methods for extracting lipids from a wet algal biomass. An exemplary method comprises lysing a wet algal biomass with an insoluble granular lysing agent to create a lysate, creating a lipid-rich phase in the lysate, and separating the lipid-rich phase from the lysate. An exemplary system comprises a lysing station for creating a lysate from a wet algal biomass and a separation station for creating and separating a lipid-rich phase from the lysate. According to further exemplary systems and methods, ultrasound may be used in place of or in addition to a lysing agent to lyse the wet algal biomass.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to extracting lipids from algal biomass, and more particularly to low energy techniques using an insoluble granular lysing agent or ultrasound to extract lipids from wet algal biomass.
2. Description of Related Art
Microalgae differentiate themselves from other single-cell microorganisms in their natural ability to accumulate large amounts of lipids. Because most lipidic compounds have the potential to generate biofuels and renewable energy, there is a need for efficient systems and methods for extracting lipids from wet algal biomass.
SUMMARY OF THE INVENTION
Presented herein are exemplary systems and methods for extracting lipids from a wet algal biomass. An exemplary method comprises lysing a wet algal biomass with an insoluble granular lysing agent to create a lysate, creating a lipid-rich phase in the lysate, and separating the lipid-rich phase from the lysate.
Exemplary methods include using an insoluble granular lysing agent having a grain size dispersion of 10 to 1000 micrometers. In some embodiments, the insoluble granular lysing agent may be sand. In other embodiments, the insoluble granular lysing agent may be chalk, gypsum, or fly ash, for example. In various embodiments, the wet algal biomass is in the presence of an extraction solvent. The extraction solvent may be monophasic and multipolar.
An exemplary system comprises a lysing station for creating a lysate from a wet algal biomass and a separation station for creating and separating a lipid-rich phase from the lysate.
According to further exemplary systems and methods, ultrasound may be used in place of or in addition to a lysing agent to lyse the wet algal biomass.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an exemplary system for extracting lipids from wet algal biomass according to one embodiment; and
FIG. 2 is a diagram showing an exemplary method for extracting lipids from wet algal biomass.



DETAILED DESCRIPTION
Microalgae produce large amounts of lipids. Lipids may be extracted from algal biomass by the use of solvents. For instance, hexane may be used to extract lipids and carotenoids from algal biomass. Acetone and carbon dioxide are other solvents that may be used to extract lipids and carotenoids from algal biomass.
Solvent extraction, however, is generally only effective if the lipid-rich biomass has a low moisture level (below approximately 12%, as measured by the content of water in the total wet algal biomass). Lipids, especially polar lipids, are partially soluble in water, which reduces the hexane extraction efficiency on wet algal biomass. Further, crushing or grinding algal cells in order to expose the intracellular biomass is generally only effective on dry algal biomass. Additionally, dewatering algal biomass by filtration is generally not very effective. Membranes, air flotation, and press, belt or drum filtration typically yield a maximum solid content of 30% (or conversely a moisture content of 70% or above). Reducing this moisture level further may be achieved by drying, which is a complicated and expensive operation that makes the production of lipids and carotenoids from algae very energy inefficient and expensive. The exemplary systems and methods described herein increase the efficiency and decrease the cost of lipid extraction from wet algal biomass.
FIG. 1 shows an exemplary system for extracting lipids from wet algal biomass according to one embodiment. The exemplary system 100 comprises a lysing/ultrasound station 105, a solid separation station 110, a two-phase liquid separation station 115, a polar aqueous phase distillation station 120, a non-polar organic phase distillation station 125, and a lipid station (not shown).
According to one embodiment, the lysing/ultrasound station 105 receives centrifuged algal biomass, polar and non-polar solvents, and a lysing agent. This mixture may be mixed, vortexed, or otherwise agitated to induce cell lysis. According to a further embodiment, cell lysing and solvent mixing take place simultaneously and continuously in a stirred tank. Agitation in the tank may be provided in different ways, including using shaking containers. The tank provides an environment where the solvents may come into effective contact with the lysed algal biomass. In an alternative embodiment, jet mixing or ultrasound may be used to agitate the lysing agent/biomass mixture.
According to an alternative embodiment, ultrasound may be used in place of the lysing agent. For example, the use of ultrasonic cell disruptor probes may be an effective cell lysis technique. A similar technique includes using a continuous flow-through ultrasonication chamber for lysing cells in an algae slurry.
In various embodiments, the solid separation station 110 receives the mixture of polar and non-polar solvents, lysing agent and lysed algal biomass. At the solid separation station 110, via centrifugation, the lysing agent, lipid-rich solvent mixture, and miscella are each isolated and directed to a different destination. The lipid-rich solvent mixture is sent to the two-phase liquid separation station 115, and the miscella is sent to a solvent recovery station (not shown). The miscella is subject to flash evaporation or similar operation to evaporate the solvents, which are subsequently condensed and recycled to the process.
The two-phase liquid separation station 115, according to one embodiment, pools the lipid-rich solvent mixture received from the solid separation station 110. The lipid-rich solvent mixture is mixed with water at the two-phase liquid separation station 115. The resulting mixture is centrifuged into two portions: an aqueous phase that includes polar solvent, and an organic phase (lipidic fraction) that includes non-polar solvent. The aqueous phase is sent to the polar aqueous phase distillation station 120 and the organic phase is sent to the non-polar organic phase distillation station 125.
At the polar aqueous phase distillation station 120, in various embodiments, the aqueous phase is distilled to recover the polar solvent. At the non-polar organic phase distillation station 125, the organic phase is distilled to recover two portions: the lipidic portion and the non-polar solvent portion. The lipidic portion is sent to the lipid station (not shown). At the lipid station, according one exemplary embodiment, the lipidic portion is heated to evaporate some or all of the remaining solvents within the lipidic portion.
FIG. 2 is a diagram showing an exemplary method 200 for extracting lipids from wet algal biomass.
As a preliminary step, wet algal biomass is centrifuged to increase its solid content to a range of approximately fifteen percent (15%) to thirty percent (30%). According to another exemplary embodiment, membrane filtration is used instead of centrifugation.
At step 210, wet algal biomass is lysed with an insoluble granular lysing agent and/or ultrasound to create a lysate. In various embodiments, the lysing agent is an insoluble granular lysing agent with a grain size dispersion of 10 to 1000 micrometers. The insoluble granular lysing agent may be sand, chalk, gypsum or fly ash. The amount of insoluble granular lysing agent used may be a quantity effective to make the intra-cellular biomass available for contact with the polar and non-polar solvents. The amount of insoluble granular lysing agent may be equivalent to the dry weight of the wet algal biomass.
According to an alternative embodiment, ultrasound may be used in place of the lysing agent. For example, the use of ultrasonic cell disrupter probes may be an effective cell lysis technique. A similar technique includes using a continuous flow-through ultrasonication chamber for lysing cells in an algae slurry.
Additionally, polar and non-polar solvents may be added to the algal biomass-lysing agent mixture to create a slurry. In one exemplary embodiment, the slurry may have a water content ranging between 10% and 90% by weight. The polar solvent may be a ketone, such as acetone, methyl-ethyl ketone or di-ethyl ketone; or an alcohol, such as methanol, ethanol, propanol, butanol, isopropanol; or an alkyl halide such as di-chloro-methane and tri-chloro-ethane; or a furane such as tetra-hydro-furane. The polar solvent, according to another exemplary embodiment, may also be dimethyl ether. In various exemplary embodiments, the non-polar solvents may include hydrocarbons such as propane, butane, pentane, hexane; ethers such as diethyl ether, diisopropyl ether, methyl tert-butyl ether; esters such as ethyl propanoate; or halocarbons such as trichloroethylene etc. Typically, the amount of polar and non-polar solvents required is proportional to the amount of water present in the biomass. In various embodiments, the mixture of polar and non-polar solvents is added in the desired solvent-extractible volume ratio. For instance, 20 to 100 volumes of solvents are usually required to extract one volume of lipids and carotenoids from algal biomass. Then, the mixture of polar and non-polar solvents, lysing agent and algal biomass is vortexed to induce cell lysis. The resulting solids are centrifuged from the vortexed mixture of polar and non-polar solvents, lysing agent and algal biomass. According to one embodiment, the lysing agent, lipid-rich solvent mixture, and miscella are each isolated.
At step 220, a lipid-rich phase is created in the lysate. According to various exemplary embodiments, the pooled lipidic fraction (less the lysing agent) is mixed with water.
At step 230, the lipid-rich phase is separated from the lysate. In one exemplary embodiment, the mixture of the lipidic fraction and water is centrifuged into two portions: an aqueous phase that includes polar solvent and an organic phase (lipidic fraction) that includes non-polar solvent. A liquid-liquid separator may be used to generate the two phases. The aqueous phase is distilled to recover the polar solvent. The organic phase is distilled to recover two portions: the lipidic portion and the non-polar solvent portion. Then the lipidic portion is heated to evaporate some or all of the remaining solvents within the lipidic portion.
EXAMPLE ONE
Fifty milliliters (50 mls) of harvested microalgae liquid culture of a density of about 300 milligrams per liter of ash-free dry biomass is centrifuged in 50 ml conical tubes at 2000 RCF for 15 minutes. The supernatant is discarded and the pellet is covered with a portion of sand, chalk, gypsum or fly ash with a volume approximately equal to that of the cell pellet. 5 ml of solvent mixture is added. The solvent may be methanol: chloroform 2:1, acetone: hexane 2:1, or any similar single phase mixture of solvents of differing polarity that form a single phase when mixed with the wet biomass and are capable of dissolving lipids.
The tube is then vortexed for five minutes to lyse the cells, and centrifuged again for 15 minutes at 2000 RCF. The supernatant is collected and fresh solvent is added to the pellet. The tube is again vortexed and centrifuged. The supernatant is collected and the pellet vortexed again with fresh solvent. After centrifugation a fourth extraction/centrifugation is performed and the 4 supernatants are combined. The lysing agent and the defatted biomass form separate layers in the centrifuge tube which may be separated by scooping the defatted biomass off the top.
The combined supernatants are centrifuged to remove any residual suspended solids. The supernatant is then mixed with 10 mls of water and the mixture is centrifuged for 5 minutes to yield a biphasic system with a dark, pigment rich non-polar solvent layer and a clear water/polar solvent layer. The layers are separated and the non-polar layer is evaporated to dryness under a stream of nitrogen to yield the microalgal lipids.
EXAMPLE TWO
50 mls of harvested microalgae liquid culture of a density between 200 and 3000 milligrams per liter of ash-free dry biomass is centrifuged in 50 ml conical tubes at 2000 RCF for 15 minutes. The supernatant is discarded and the pellet is exposed to a 20 kHz ultrasonic pulse for 3 minutes from a Branson 450 Sonifier. 5 ml of solvent mixture is added. The solvent may be methanol:chloroform 2:1, acetone hexane 2:1, or any similar single phase mixture of solvents of differing polarity that form a single phase when mixed with the wet biomass and are capable of dissolving lipids. The tube is then vortexed for 5 minutes to mix the lysed cells with the solvent, and centrifuged again for 15 minutes at 2000 RCF. The supernatant is collected and fresh solvent is added to the pellet. The tube is again vortexed and centrifuged. The supernatant is collected and the pellet vortexed again with fresh solvent. After centrifugation a fourth extraction/centrifugation is performed and the 4 supernatants are combined. The combined supernatants are centrifuged to remove any residual suspended solids. The supernatant is then mixed with 10 mls of water and the mixture is centrifuged for 5 minutes to yield a biphasic system with a dark, pigment rich non-polar solvent layer and a clear water/polar solvent layer. The layers are separated and the non-polar layer is evaporated to dryness under a stream of nitrogen to yield the microalgal oils.
While various embodiments have been described herein, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of a preferred embodiment should not be limited by any of the herein-described exemplary embodiments.

## Claims

1. A method comprising:
lysing a wet algal biomass with an insoluble granular lysing agent to create a lysate;
creating a lipid-rich phase in the lysate; and
separating the lipid-rich phase from the lysate.

2. The method of claim 1, wherein the insoluble granular lysing agent has a grain size dispersion of 10 to 1000 micrometers.

3. The method of claim 1, wherein the insoluble granular lysing agent is sand.

4. The method of claim 1, wherein the insoluble granular lysing agent is chalk.

5. The method of claim 1, wherein the insoluble granular lysing agent is gypsum.

6. The method of claim 1, wherein the insoluble granular lysing agent is fly ash.

7. The method of claim 1, wherein the wet algal biomass is in the presence of an extraction solvent.

8. The method of claim 7, wherein the extraction solvent is monophasic and multipolar.