WO2010123903A1

WO2010123903A1 - Systems, apparatus and methods for obtaining intracellular products and cellular mass and debris from algae and derivative products and process of use thereof

Info

Publication number: WO2010123903A1
Application number: PCT/US2010/031756
Authority: WO
Inventors: Nicholas D. Eckelberry; Michael Philip Green; Scott Alexander Fraser
Original assignee: Originoil, Inc.
Priority date: 2009-04-20
Filing date: 2010-04-20
Publication date: 2010-10-28
Also published as: CN102449155B; EP2421983A1; BRPI1013863A2; AU2010239380B2; JP2012523849A; JP5284536B2; AU2010239380A1; CN102449155A; MX2011011035A

Abstract

Systems and methods for harvesting at least one intracellular product (e.g., lipids, carbohydrates, proteins, etc.) from algae cells in aqueous suspension and for harvesting a mass of ruptured algae cells and debris from an aqueous solution containing algae cells make use of an apparatus that includes an electrical circuit. The electrical circuit includes an outer anode structure (e.g., tube) which provides containment for an inner structure (e.g., electrical conductor) having lesser dimensions than the outer anode structure, the inner structure serving as a cathode. A spiraling surface, such as a plurality of grooves separated by at least one land, much as in the nature of "rifling" in the barrel of a gun, or alternatively, an electrically insulative, isolator spacer in parallel to both structures (e.g., the outer tube and internal conductor) provides a liquid seal and provides spacing between the anode and cathode circuits which is required for equal electrical distribution and to prevent short circuiting of the flow path for the aqueous solution containing algae cells.

Description

SYSTEMS, APPARATUS AND METHODS FOR OBTAINING INTRACELLULAR PRODUCTS AND CELLULAR MASS AND DEBRIS FROM

ALGAE AND DERIVATIVE PRODUCTS AND PROCESS OF USE THEREOF

FIELD OF THE INVENTION

[0001] The invention relates to the fields of energy and microbiology. In particular, the invention relates to systems, apparatus and methods for harvesting cellular mass and debris as well as intracellular products from algae cells which can be used as a substitute for fossil oil derivatives in various types of product manufacturing.

BACKGROUND OF INVENTION

[0002] The intracellular products of microorganisms show promise as a partial or full substitute for fossil oil derivatives or other chemicals used in manufacturing products such as pharmaceuticals, cosmetics, industrial products, biofuels, synthetic oils, animal feed, and fertilizers. However, for these substitutes to become viable, methods for obtaining and processing such intracellular products must be efficient and cost-effective in order to be competitive with the refining costs associated with fossil oil derivatives. Current extraction methods used for harvesting intracellular products for use as fossil oil substitutes are laborious and yield low net energy gains, rendering them unviable for today's alternative energy demands. Such methods can produce a significant carbon footprint, exacerbating global warming and other environmental issues. These methods, when further scaled up, produce an even greater efficiency loss due to valuable intracellular component degradation and require greater energy or chemical inputs then what is currently financially feasible from a microorganism harvest. For example, the cost per gallon for microorganism bio-fuel is currently approximately nine-fold over the cost of fossil fuel.

[0003] Recovery of intracellular particulate substances or products from microorganisms requires disruption or lysing of the cell transmembrane. All living cells, prokaryotic and eukaryotic, have a plasma transmembrane that encloses their internal contents and serves as a semi-porous barrier to the outside environment. The transmembrane acts as a boundary, holding the cell constituents together, and keeps foreign substances from entering. According to the accepted current theory known as the fluid mosaic model (SJ. Singer and G. Nicolson, 1972), the plasma membrane is composed of a double layer (bi-layer) of lipids, an oily or waxy substance found in all cells. Most of the lipids in the bilayer can be more precisely described as phospholipids, that is, lipids that feature a phosphate group at one end of each molecule. [0004] Within the phospholipid bilayer of the plasma membrane, many diverse, useful proteins are embedded while other types of mineral proteins simply adhere to the surfaces of the bilayer. Some of these proteins, primarily those that are at least partially exposed on the external side of the membrane, have carbohydrates attached and therefore are referred to as glycoproteins. The positioning of the proteins along the internal plasma membrane is related in part to the organization of the filaments that comprise the cytoskeleton, which helps anchor them in place. This arrangement of proteins also involves the hydrophobic and hydrophilic regions of the cell. [0005] Intracellular extraction methods can vary greatly depending on the type of organism involved, their desired internal component(s), and their purity levels. However, once the cell has been fractured, these useful components are released and typically suspended within a liquid medium which is used to house a living microorganism biomass, making harvesting these useful substances difficult or energy-intensive.

[0006] In most current methods of harvesting intracellular products from algae, a dewatering process has to be implemented in order to separate and harvest useful components from a liquid medium or from biomass waste (cellular mass and debris). Current processes are inefficient due to required time frames for liquid evaporation or energy inputs required for drying out a liquid medium or chemical inputs needed for a substance separation.

[0007] Accordingly, mere is a need for a simple and efficient procedure for harvesting intracellular products from microorganisms that can be used as competitively-priced substitutes for fossil oils and fossil oil derivatives required for manufacturing of industrial products.

SUMMARY OF THE INVENTION

[0008] Described herein are systems, methods and apparatuses for harvesting at least one intracellular product from algae cells in aqueous suspension and for harvesting cellular mass and debris from an aqueous solution containing algae cells. The systems and methods make use of an apparatus that includes an electrical circuit. The electrical circuit includes an outer anode structure (e.g., tube) which provides containment for an inner structure (e.g., electrical conductor) having lesser dimensions than the outer anode structure, the inner structure serving as a cathode. A spiraling surface, such as a plurality of grooves separated by at least one land, much as in the nature of "rifling" in the barrel of a gun, or alternatively, an electrically insulative, isolator spacer in parallel to both structures (e.g., the outer tube and internal conductor), provides a liquid seal and provides spacing between the anode and cathode circuits which is required for equal electrical distribution and to prevent short circuiting of the flow path for the aqueous solution containing algae cells.

[0009] The outer anode structure (e.g., tube) typically includes a pair of containment sealing end caps with one end cap having an entry provision used to accept an incoming flow of microorganism biomass, referred to herein as a live slurry or aqueous suspension including microorganism cells, and an opposing end cap through which the transiting flow of biomass exits. The inner cathode structure (e.g., electrical conductor which may optionally also be a tube of the same or different shape as the outer tube) also typically includes sealed end caps to disallow a liquid flow through the center of the structure (e.g., the inner tube) and to divert the flow in between the wall surfaces of the anode and cathode circuits.

[0010] A spiraling isolator spacer serves as a liquid seal between the two wall surfaces of the electrical conductors and with the thickness of the spacer preferably providing equal distance spacing between the two individual wall surfaces. Spacing should be considered critical for allowing a complete three hundred and sixty degree transfer of electrical current around each circuit assembly and the prevention of a short circuit by touching of the anode and cathode surfaces. Further, the spiraling isolator now provides a gap between the two wall surfaces allowing a passage way for a flowing biomass. The spiraling directional flow provided by the spiraling isolator or rifling also provides longer transit duration for greater electrical exposure to the flowing biomass thus increasing substance extraction efficiency and allowing a lower watt per hour consumption rate when the circuit is scaled up in size for large volume flows. [0011] Pulse frequency transfer should be conducted on the negative side of the circuit thus being transmitted through the anode with negative transfer to the cathode. This method allows a greater efficiency in electrical energy transfer between the anode and cathode surfaces, [0012] Due to cellular magnetic polarities, a magnetic response occurs once subject cells transit through the circuit. Magnetic cellular alignment is due to their respective positive and negative polarities when exposed to the concurring electromagnetic field generated during the electrical on pulse phases. After cellular alignment, the electromagnetic field continues to create a pulling force on the cells while they absorb the electrical current in a way similar to an electrical capacitor storing voltage. This causes the cell's intracellular components to swell and weaken the cellular wall structure to the point of no longer being capable of containing its intracellular components. At the point of maximum expansion pressure, a total collapse of the outer cellular wall structure occurs allowing the release of all internal cell components. [0013] Electrical input frequency rates should be determined by biomass density with pulse rate frequencies increased when a thicker biomass is present. Biomass density is determined by using a formula based on a percentage of grams of biomass present per liter of flowing liquid medium.

[0014] Use of this formula allows a programmable microprocessor working in conjunction with a series of sensors to assume operational responsibilities. Based on biomass density formulas, an automated matrix dictates to the system the prescribed parameters for flow, the amount of electrical input and the rate of frequency required for efficient substance extraction. This practice further allows greater energy efficiency in larger scale applications. [0015] Accordingly, described herein is an apparatus for harvesting at least one intracellular component from algae cells in aqueous suspension. The apparatus includes: at least one first electrical conductor that acts as a cathode and a second electrically conductive housing that acts as an anode, the at least one first conductor being disposed within the housing, such that a space is defined between the exterior of the first conductor and an interior of the housing, providing a flow path for the aqueous suspension, wherein at least a portion of one or both surfaces of the first conductor and the housing has been removed to create at least two spiral grooves separated by at least one land that reduces or prevents algae cell buildup on or around the first conductor and the housing; an electrical power source operably connected to the first conductor and the housing for providing a pulsed electrical current that is applied between the first conductor and the housing and the aqueous suspension for rupturing the algae cells resulting in a mass of ruptured algae cells (cellular mass and debris) and release of intracellular components from the algae cells in the aqueous suspension; and a secondary tank that is operably connected to the first electrical conductor and the housing such that the aqueous suspension can flow from the flow path into the secondary tank for separation of the at least one intracellular component from the algae cells in aqueous suspension. In the apparatus, the first conductor can be a metal tube. The first conductor and second housing can each be metal tubes, e.g., metal tubes of circular shape, metal tubes of different shapes, etc. In one embodiment, the inner diameter of the metal housing and the outer diameter of the first conductor differ in size on the order of 0.050 inch. In the apparatus, the housing can be a metal tube and the at least one electrical conductor can include a plurality of spaced apart electrical conductors, the electrical conductors being separated from each other by electrically insulating elements; and a multiplicity of flow paths being created between the housing and each of the plurality of spaced apart electrical conductors, In this embodiment, each of the plurality of electrical conductors can be metal tubes. [0016] Also described herein is a method of harvesting at least one intracellular component from algae cells in aqueous suspension. The method includes providing an apparatus that includes: at least one first electrical conductor that acts as a cathode and a second electrically conductive housing thai acts as an anode, the at least one first conductor being disposed within the housing, such that a space is defined between the exterior of the first conductor and an interior of the housing, providing a flow path for the aqueous suspension, wherein at least a portion of one or both surfaces of the first conductor and the housing has been removed to create at least two spiral grooves separated by at least one land that reduces or prevents algae cell buildup on or around the first conductor and the housing; an electrical power source operably connected to the first conductor and the housing for providing a pulsed electrical current that is applied between the first conductor and the housing and the aqueous suspension for rupturing the algae cells resulting in a mass of ruptured algae cells (cellular mass and debris) and release of intracellular components from the algae cells in the aqueous suspension; a secondary tank that is operably connected to the first electrical conductor and the housing such that the aqueous suspension can flow from the flow path into the secondary tank for separation of the at least one intracellular component from the algae cells in aqueous suspension; and an aqueous suspension including conductive minerals and algae cells wherein the aqueous suspension is disposed in the flow path of the apparatus. The method further includes the steps of: applying a sufficient amount of a pulsed electrical current to the at least one first conductor and the housing and aqueous suspension for caused alternature expansion and contraction of the cell contents thereby rupturing the algae cells resulting in a mass of ruptured algae cells (cellular mass and debris) and release of intracellular components from the algae cells in the aqueous suspension; flowing the aqueous suspension containing the mass (cellular mass and debris) and released intracellular components to the secondary tank for separating the intracellular components from the cellular mass and debris and aqueous suspension; and separating the at least one intracellular component from the cellular mass and debris and aqueous suspension,

[0017] Further described herein is a method of harvesting cellular mass and debris from an aqueous suspension including algae cells. The method includes providing an apparatus that includes: at least one first electrical conductor that acts as a cathode and a second electrically conductive housing that acts as an anode, the at least one first conductor being disposed within the housing, such that a space is defined between the exterior of the first conductor and an interior of the housing, providing a flow path for the aqueous suspension, wherein at least a portion of one or both surfaces of the first conductor and the housing has been removed to create at least two spiral grooves separated by at least one land that reduces or prevents algae cell buildup on or around the first conductor and the housing; an electrical power source operably connected to the first conductor and the housing for providing a pulsed electrical current that is applied between the first conductor and the housing and the aqueous suspension for rupturing the algae cells resulting in a mass of ruptured algae cells (cellular mass and debris) and release of intracellular components from the algae cells in the aqueous suspension; a secondary tank that is operably connected to the first electrical conductor and the housing such that the aqueous suspension can flow from the flow path into the secondary tank for separation of the at least one intracellular component from the algae cells in aqueous suspension; an element disposed in the secondary tank for producing microbubbles; an aqueous suspension including conductive minerals and algae cells wherein the aqueous suspension is disposed in the flow path of the apparatus; and a pump disposed in the secondary tank for circulating the aqueous suspension. The method further includes the steps of: applying a sufficient amount of a pulsed electrical current to the at least one first conductor and the housing and aqueous suspension for rupturing the algae cells resulting in release of intracellular components from the ruptured algae cells and a mass of ruptured algae cells (cellular mass and debris) in the aqueous suspension; flowing the aqueous suspension containing the released intracellular components and cellular mass and debris to the secondary tank for separating the cellular mass and debris from the released intracellular components and the aqueous suspension; activating the pump and the element for producing microbubbles resulting in a plurality of microbubbles that attach to the released intracellular components and float upwards in the aqueous suspension and the sinking of the cellular mass and debris downwards in the aqueous suspension; and separating the cellular mass and debris from the released intracellular components and aqueous suspension. The element disposed in the secondary tank for producing microbubbles can be any suitable device or apparatus, e.g. a mixer,

[0018] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0019] As used herein, the phrases "intracellular products" and "intracellular products from algae cells" refer to any molecule, compound or substance found within an algae cell. Examples of intracellular products from algae cells include lipids, proteins, carbohydrates (e.g., glucose), carotenoids, nucleic acids, hydrogen gas, etc.

[0020] By the term "biomass" is meant unicellular organisms and single cell organisms grown in a liquid medium for the purpose of harvesting intra cellular components such as triglycerides, proteins or carbohydrates.

[0021] As used herein, the phrase "cellular mass and debris" means the products that result from rupturing of a cell.

[0022] As used herein, the term "live slurry" relates to the biomass as defined above in a state of growth within a matrix such as salt water, waste water or fresh water. "Biomass" and

"live slurry" are used interchangeably herein.

[0023] Although methods, systems and apparatus similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods, systems and apparatus are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1 A and IB schematically depict a pair of flow diagrams illustrating a method of harvesting at least one intracellular product from algae cells in aqueous suspension as described herein (referred to as "single step extraction") (FIG. IA) and a method of harvesting cellular mass and debris from an aqueous solution containing algae cells as described herein (referred to herein as "single step extraction & quantum fracturing") (FIG. IB).

[0025] FIG. 2 illustrates a sectional perspective view of biomass flowing in between the anode and cathode wall surfaces and the electrical transfer circuit of one embodiment of an apparatus as described herein.

[0026] FlG. 3 illustrates a perspective view of the inner and outer end caps in location on the anode and cathode tubes of one embodiment of an apparatus as described herein.

[0027] FIG. 4 ill ustrates a perspective sectional view of the spiral spacer in between the anode and cathode tubes of one embodiment of an apparatus as described herein.

[0028] FIG. 5 is a perspective view of a series of anode and cathode circuits connected in parallel by an upper and lower manifold of one embodiment of an apparatus as described herein.

[0029] FIG. 6 illustrates an EMP apparatus as described herein with a flowing liquid medium containing a microorganism biomass being exposed to an electromagnetic field caused by an electrical transfer.

[0030] FIG. 7 illustrates an EMP apparatus as described herein, directional flowing biomass with applied heat being absorbed and transferred into the liquid medium.

[0031] FIG. 8 illustrates an overview of a normal sized microorganism cell in relationship to a secondary illustration of a swollen cell during exposure to an electromagnetic field and electrical charge.

[0032] FIG. 9 Illustrates a side view of a micron mixer in association with a secondary tank containing a biomass and sequences of developing foam layers generated by a micron mixer.

[0033] FlG. 10 illustrates a secondary tank containing the liquid medium and a resulting foam layer capable of being skimmed off the surface of the liquid medium, into a foam harvest tank.

[0034] FIG. 11 illustrates one embodiment of a method and apparatus (system) as described herein for the harvest of useful substances from an algae biomass involving single step extraction,

[0035] FIG. 12 illustrates another embodiment of a method and apparatus (system) as described herein for the harvest of useful substances from an algae biomass involving single step extraction.

[0036] FIG. 13 illustrates an example of a modified static mixer. [0037 j FIG. 14 is a table of data from experiments to quantify lipid extraction and identify optimal extraction parameters.

DETAILED DESCRIPTION

[0038] Described herein are systems, methods and apparatuses for harvesting at least one intracellular product from algae cells in aqueous suspension and for harvesting cellular mass and debris from an aqueous solution containing algae cells. These systems, methods and apparatus involve subjecting algae cells to a pulsed electrical current (an EMP) based on the algae cells' ability to be magnetically responsive and electrically conductive due to the uptake of nutrients required for their survival. Most of these nutrients contain conductive minerals and when digested, are retained within the cells' transmembranes. Most aquatic microorganism cells consist of a transmembrane which houses the internal membrane components such as the nucleus, chloroplast, proteins, and lipids and with most internal regions surrounded by an internal liquid mass. Due to cellular composition, when exposed to electrical current, intracellular components expand in size due to electrical adsorption. However, during an off electrical phase, intracellular components immediately contract back down in size. When electrical current is pulsed in rapid frequency, intracellular components and their surrounding liquid mass undergo rapid rates of expansion and contraction. Due to expansion pressures, the rapid on and off frequency produces an internal pounding pressure against the transmembrane, resulting in eventual fracturing. Once fractured, ongoing electrical frequency continues the on and off pressure build-up due to the surrounding liquid mass which aids in the extrusion or expulsion of internal cellular components outside of the transmembrane boundaries. The below described preferred embodiments illustrate adaptations of these systems, apparatuses and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

[0039] A typical method of harvesting at least one intracellular product from algal cells (referred to herein as "single step extraction," see FlG. IA) includes subjecting algae cells in aqueous suspension to an EMP in an apparatus as described herein, resulting in rupturing of the algae cells and separation of intracellular lipids (or other intracellular products) from the resultant cellular mass and debris. In a typical bench scale EMP application, an electrical current of 1 - 60 peak amps @ I-24volts or 25w to 500 watts is applied. For example, at 1 gallons per minute (GPM) of throughput with a culture having a density of 500 mg/L, one would use approximately 70 watts of energy (3.5v @ 20 peak amps) for a successful extraction. At 5 GPM, the same culture would require approximately 350 watts (3.5v @ 100 peak amps). In this method, the algae cells in aqueous suspension can optionally be subjected to heat which can increase cell rupturing, improving harvesting efficiency by about 20-50%. Heat can be applied to the cells before (upstream of) the EMP, or heat can be applied to the cells in the apparatus {e.g., concomitantly with EMP). A method of harvesting cellular mass and debris from an aqueous solution containing algae cells (referred to herein as "single step extraction plus quantum fracturing," see FIG. IB) includes subjecting algae cells to EMP and to cavitation (i.e., microbubbles) in an apparatus as described herein, resulting in a mixture that includes both intracellular product(s) (e.g., lipids) and cellular mass and debris. The cells can be subjected to cavitation before application of (upstream of) EMP, or they may be subjected to cavitation concomitantly with EMP (see FIG. 13 that depicts the cavitation device electrified as it would be the BMP conductor). In one embodiment, a cavitation device includes an anode, cathode and venture mixer (all in one). In this embodiment, the cavitation unit is reduced (e.g., by half), a non-conductive gasket is added, and it is electrified. Under normal pressure conditions, e.g., under 100 psi, no effect was observed when cavitation was applied upstream of EMP, however, at pressures above 100 psi (e.g., 110, 115, 120, 130, 140, 150, 200, 300, 400 psi, etc.), it may have an effect. In a method of harvesting cellular mass and debris from an aqueous solution containing algae cells, the algae cells in aqueous suspension can optionally be subjected to heat to achieve sinking of the cellular mass and debris and rising of intracellular products (e.g., lipids) within the apparatus, thereby facilitating separation of the intracellular products from the cellular mass and debris. Heat can be applied to the culture (containing the cells) before (upstream of) the EMP, or heat can be applied to the culture in the apparatus (e.g., concomitantly with EMP as shown in FIG. 13). In a typical method, e.g., at .5 GPM, 500 mg/L density, an electrical current of approximately 60 watts (15 peak amps @ 4 volts) is applied. Generally, a GPM of approximately .1 to approximately 5 GPM and watts in the range of about 20 to about 1000 watts (e.g., 2-18volts @ 2-50 peak amps) are used. For example, at 1 GPM of throughput with a culture having a density of 500 mg/L, one could use approximately 70 watts of energy (3.5v @ 20 peak amps) for a successful extraction. At 5 GPM, the same culture would require approximately 350 watts (3.5v @ 100 peak amps).

[0040] An apparatus as described herein for harvesting at least one intracellular product from algae cells in aqueous suspension or for harvesting cellular mass and debris from an aqueous solution containing algae cells includes a flow path between two metal surfaces, such as the flow path created between two metal plates of large surface area, separated by a small distance. In a typical embodiment, the flow path is created in an annulus created between an inner metallic surface of a tube and an outer surface of a smaller metallic conductor placed in the tube. The tubes need not have a circular periphery as an inner or outer tube may be square, rectangular, or other shape and the tube shape does not necessarily have to be the same, thereby permitting tube shapes of the inner and outer tubes to be different. In a most preferred embodiment, the inner conductor and outer tube are concentric tubes, with at least one tube, preferably the outer tube, being provided with a plurality of spiral grooves separated by lands to impart a rifling to the tube. Tliis rilling has been found to decrease build-up of residue on the tube surfaces. In commercial production, there may be a plurality of inner tubes surrounded by an outer tube to increase the surface contact of the metal conductors with the algae containing vehicle, such as the culture to impart high electrical transfer through the culture (brine would apply to salt water algae, but the device can successfully process fresh water algae as well) to the algae cells contained therein. Furthermore, the use of electrical insulators, such as plastic tubes, baffles, and other devices, can be used to separate a large EMP apparatus into a plurality of zones, so as to efficiently scale-up the invention to commercial applications. The systems, methods and apparatuses for harvesting at least one intracellular product from algae cells in aqueous suspension and for harvesting cellular mass and debris from an aqueous solution containing algae cells can be applied to any algae cell. In the experiments described below, Nanochloropsis oculata cells were used. However, intracellular products can be obtained from any algae cells. Examples of additional algae cells include Scenedesmus, Chlamydomonas, Chloreila, Spirogyra, Englena, Prymneshim, Porphyridium, Synechoccus sp, Cyanobacteria and certain classes of Rhodophyta single celled strains. The cells can be grown and applied to an apparatus as described herein at any suitable concentration, e.g., from about 100 mg/L to about 5 g/1 (e.g., about 500 mg/L to about 1 g/L), Cell concentrations of about 500 mg/L and about 1 mg/L have been successfully used. In some embodiments, unconcentrated algae from a growth vessel will be from 250 mg/L to 1.5 g/L and may be pre-concentrated with other conventional means from 5 g/L up to 20 g/L.

[0041] Referring to FlG. 2, an apparatus 22 as described herein for harvesting at least one intracellular product from algae cells in aqueous suspension or for harvesting cellular mass and debris from an aqueous solution containing algae cells is shown. A liquid containing a living microorganism biomass, 1 is flowed in between the inside wall surface of the anode tube, 2 and the outside wall surface of an inner cathode tube 3. By way of an electrical conduit, a negative connection 4 is made to the anode tube 2 which provides electrical grounding transfer of the entire tube. Positive electrical input 5 also delivered by way of a conduit connection provide positive electrical transfer throughout the cathode tube 3.

[0042] When a positive current 5 is applied to the cathode 3 it then seeks a grounding circuit for electrical transfer 6 or in this case, to the anode 2 which allows the completion of the electrical circuit, In this respect, transfer of electrons occurs between the positive and negative surfaces areas but only when an electrically conductive liquid is present between them. As the liquid medium containing a living microorganism biomass 1 is flowed between the surface areas, electrical transfer from the cathode tube 3 through the liquid 1 to the anode tube 2 is made. As a liquid containing a microorganism biomass transverses the anode and cathode circuit, the cells are exposed to both a magnetic field, causing a cellular alignment, and to an electrical field which induces cellular current absorption.

[0043] In reference to FIG. 3, the outer anode tube 2 requires a pair of containment sealing end caps 7 and 8. Sealing end cap 7 provides an entry point 9 used to accept a flowing microorganism biomass. After biomass transiting, the opposing end cap 8 provides an exit point 10 to the outward flowing biomass.

[0044] As shown also in FIG. 3, the inner cathode tube 3 as well requires sealed end caps 1 1 and 12 to disallow a liquid flow through the center of the tube and to divert the flow in between the wall surfaces of the anode and cathode.

[0045] In reference to FIG. 4 an electrically insulative spiraling isolator spacer 13 serves as a liquid seal between the two wall surfaces 14 and 15 with the thickness of the spacer preferably providing equal distance spacing between the anode 2 and the cathode 3. Spacing is important for allowing a complete three hundred and sixty degree transfer of electrical current around the anode 2 and cathode tube 3 as contact between the anode 2 and cathode 3 will create a short circuit impairing electrical transfer through the liquid medium. Further the spiralmg isolator 13 now provides a gap 16 between the two wall surfaces 14 and 15 allowing a passage way for a flowing biomass 1. The spiraling directional flow further provides a longer transit duration which provides greater electrical exposure to the flowing biomass 1 thus increasing substance extraction efficiency at a lower per kilowatt hour consumption rate during intracellular substance extraction. Any suitable material can be used as a spacer. Typically, ceramic, polymeric, vinyl, PVC plastics, bio-plastics, vinyl, monofilament, vinyl rubber, synthetic rubber, or other non- conductive materials are used.

[0046] In reference to FlG. 5, a series of anode and cathode circuits 17 are shown in parallel having a common upper manifold chamber 18 which receives an in flowing biomass 1 through entry port 20. Once entering into the upper manifold chamber 18, the biomass I makes a downward connection into each individual anode and cathode circuit 17 through entry ports 9 which allow a flowing connection to the sealing end caps 8. It is at this point where the flowing biomass 1 enters into the anode and cathode circuits 17. Once transiting in spiral through the individual circuits 17, the flowing biomass 1 exits into a lower manifold chamber 19 where the biomass 1 is then directed to flow out of the apparatus 22 (system) through exit point 21. [0047] In a method of harvesting at least one intracellular product from algae cells in aqueous suspension, the cells are grown in a growth chamber. A growth chamber (also referred to herein as a "reactor") can be any body of water or container or vessel in which all requirements for sustaining life of the algae cells are provided for. Examples of growth chambers include an open pond or an enclosed growth tank. The growth chamber is operably connected to an apparatus 22 as described herein such that algae cells within the growth chamber can be transferred to the apparatus 22, e.g., by way of gravity or a liquid pump, the living bio mass is flowed via a conduit into the inlet section of the anode and cathode circuit. Algae cells within the growth chamber can be transferred to the apparatus 22 by any suitable device or apparatus, e.g., pipes, canals, or other conventional water moving apparatus, hi order to harvest at least one intracellular product from the algae cells, the algae cells are moved from the growth chamber to an apparatus 22 such as those shown in FIGS. 2-12 as described above, and contained within the apparatus 22, When added to the apparatus 22, the algae cells are generally in the form of a live slurry (also referred to herein as "biomass"). The live slurry is an aqueous suspension that includes algae cells, water and nutrients such as an algal culture formula based on Guillard's 1975 F/2 algae food formula that provides nitrogen, vitamins and essential trace minerals for improved growth rates in freshwater and marine algae. Any suitable concentration of algae cells and sodium chloride, fresh, brackish or waste water can be used, such that the algae cells grow in the aqueous suspension.

[0048] After the algae cells are ruptured in the apparatus 22, they are then subjected to one or more downstream treatments including gravity clarification (see FIG. IA). Gravity clarification generally occurs in a clarification tank in which the intracellular product(s) of interest (e.g., lipids) rises to the top of the tank, and the cellular mass and debris sinks to the bottom of the tank. In such an embodiment, upon transiting the circuit, the fractured cellular mass and debris is flowed over into a gravity clarification tank that is operably connected to an apparatus 22 for harvesting cellular mass and debris and intracellular products from algae cells as described herein. In the gravity clarification tank, the lighter, less dense substances float to the top of the liquid column while the heavier, denser remains sink to the bottom for additional substance harvest.

[0049] The intracellular product(s) of interest is then easily harvested from the top of the tank such as by skimming or passing over a weir, and the cellular mass and debris can be discarded, recovered and/or further processed. A skimming device then can be used to harvest the lighter substances floating on the surface of the liquid column while the heavier cellular mass and debris remains can be harvested from the bottom of the clarification tank. The remaining liquid (e.g., water) can be filtered and returned to the growth chamber (recycled) or removed from the system (discarded). In an embodiment in which the intracellular product is oil (i.e., lipids), the oil can be processed into a wide range of products including vegetable oil, refined fuels (e.g., gasoline, diesel, jet fuel, heating oil), specialty chemicals, nutraceuticals, and pharmaceuticals, or biodiesel by the addition of alcohol. Intracellular products of interest can be harvested at any appropriate time, including, for example, daily (batch harvesting). In another example, intracellular products are harvested continuously (e.g., a slow, constant harvest). The cellular mass and debris can also be processed into a wide range of products, including biogas (e.g., methane, synthetic gas), liquid fuels (jet fuel, diesel), alcohols (e.g., ethanol, methanol), food, animal feed, and fertilizer.

[0050] In addition to gravity clarification, any suitable downstream treatment can be used. Possible downstream treatments are numerous and may be employed depending on the desired output/use of the intracellular contents and/or bio cellular mass and debris mass. For example, lipids can be filtered by mechanical filters, centrifuge, or other separation device, for example, then heated to evacuate more water. The lipids can then be further subjected to a hexane distillation, In another example, cellular mass arid debris can be subjected to an anaerobic digester, a steam dryer, or belt press for additional drying for food, fertilizer etc. As shown in FIG. IAj downstream treatments also include, e.g., polishing and gravity thickening, [0051] As described above, a method of harvesting cellular mass and debris from an aqueous solution containing algae cells (single step extraction plus quantum fracturing) includes subjecting algae cells to EMP and to cavitation (i.e., microbubbles) in an apparatus as described herein, resulting in a mixture that includes both intracellular product(s) (e.g., lipids) and cellular mass and debris. As with a method of harvesting at least one intracellular product from algae cells in aqueous suspension, a method of harvesting cellular mass and debris from an aqueous solution containing algae cells involves an EMP generated by an electrical transfer that is utilized for energy transfer through a liquid medium containing a living microorganism biomass (the slurry, or living slurry or aqueous suspension). This transfer is achievable due to nutrients containing electrically conductive minerals suspended within the liquid medium. An example of a typical mineral formulation is Guillϊard's 1957 formula (.82% Iron, 0.034% Manganese, 0.002% Cobalt, 0.0037% Zinc, 0.0017% Copper, 0.0009% Molybdate, 9.33% Nitrogen, 2.0% Phosphate, 0.07% Vitamin Bl, 0.0002% Vitamin B12, and 0.0002% Biotin). These nutrients are also required and consumed by a microorganism biomass in order to sustain biomass cell growth and reproduction and like the liquid medium, the consumed minerals allow the microorganism bio mass to be electrically conductive and magnetic-responsive. [0052] In the method, a micron mixing device, such as a static mixer or other suitable device such as a high throughput stirrer, blade mixer or other mixing device is used to produce a foam layer composed of microbubbles within a liquid medium containing a previously lysed microorganism biomass. Any device suitable for generating microbubbles, however, can be used. Following micronization, the homogenized mixture begins to rise and float upwards. As this mixture passes upwards through the liquid column, the less dense valuable intracellular substances freely attach to the rising bubbles, or due to bubble collision, into a heavier sinking cellular mass and debris waste, (now allowed to sink due to heated water specifics). The rising bubbles also shake loose trapped valued substances (e.g., lipids) which also freely adhere to the rising bubble column. Once the foam layer containing these useful substances has risen to the top of the liquid column, they now can be easily skimmed from the surface of the liquid medium and deposited into a harvest tank for later product refinement. Once the foam layer rises to the top of the secondary tank, the water content trapped within the foam layer generally results in less than 10% (e.g., 5, 6, 7, 8, 9, 10, 10.5, 11 %) of the original liquid mass. Trapped within the foam are the less dense useful substances, and the foam is easily floated or skimmed off the surface of the liquid medium. This process requires only dewatering of the foam, rather than evaporating the total liquid volume needed for conventional harvest purposes. This drastically reduces the dewatering process, energy or any chemical inputs while increasing harvest yield and efficiency as well as purity. In this method, water can be recycled to the growth chamber or removed from the system. Cellular mass and debris can be harvested at any appropriate time, including, for example, daily (batch harvesting). In another example, cellular mass and debris is harvested continuously (e.g., a slow, constant harvest),

[0053] In a method and apparatus for harvesting cellular mass and debris from an aqueous solution containing algae cells (single step extraction plus quantum fracturing) as described herein, a heating process can be applied during the EMP process in order to change the specific gravity of the liquid medium (the specific gravity of water density is optimal at 40 degrees F). As the liquid medium (typically mainly composed of water) is heated, alterations to its hydrogen density occurs; this alteration of density allows a normally less dense material to sink or in this case, heavier fractured cellular mass and debris materials which would normally float, now rapidly sink to the bottom of the liquid column. This alteration also allows easier harvesting of these materials which are also useful for other product applications. Once the EMP and heating process has been achieved, the liquid medium containing a now fractured biomass is transferred into a secondary holding tank where a liquid pump allows a continuous loop flow. As used in this description "specific gravity" is a dimensionless unit defined as the ratio of density to a specific material as opposed to the density of the water at a specified temperature. [0054] In one example of a method and apparatus of harvesting cellular mass and debris from an aqueous solution containing algae cells (single step extraction plus quantum fracturing) as described herein, an electrical pulse is repeated in frequencyto create an electromagnetic field and electrical energy transfer between two electrically conductive metal pieces when a conductive liquid, medium containing a living microorganism biomass is flowed between them. As this pulsed electrical transfer occurs, an electromagnetic field is produced resulting in the elongation of the bioraass cells due to their polarity. Further, the suspended biomass absorbs electrical input which causes internal cellular components and their liquid mass to swell in size. Due to swelling, an internal pressure is applied against the transmembrane, however this internal swelling is to be considered as only momentary as it is relieved during an off frequency phase of the pulsed electrical input. Rapid repeating of the on and off electrical frequency eventually weakens the elongated cells and assists in the fracturing of their transmembranes. Continuous frequency inputs further produce internal pressures caused by expanded internal component swelling which eventually forces leakage of internal phospholipid substrates to escape their outer fractured boundaries and into the liquid medium through the differential of osmotic pressures on the cell wall.

[0055] Additionally, for greater efficiency, the amount of electrical or frequency input can be adjusted based on a matrix formula of grams of biomass contained in 1 liter of the liquid medium.

[0056] Once the liquid medium has achieved passage through the EMP apparatus, it is allowed to flow over into a secondary tank (or directly into a device that is located near the bottom of the tank). Ln this method of dewatering, the secondary tank is a tank containing a micron bubble device or having a micron bubble device attached for desired intracellular component separation and dewatering. After transmembrane lysis, a static mixer or other suitable device (e.g., any static mixer or device which accomplishes a similar effect producing micro -bubbles) is used and is located at the lowest point within a secondary tank. When activated, the static mixer produces a series of micron bubbles resulting in a foam layer to develop within the liquid medium. As the liquid medium is continuously pumped through the micro mixer, bubbled foam layers radiate outwards through the liquid and begin to rise and float upwards. The less dense desired intracellular components suspended within the liquid medium attach to the micron bubbles floating upwards and flocculate to the surface or are detached from heavier sinking biomass waste, (allowed to sink due to specific gravity alterations) due to rising bubble collision within the water column.

[0057] In reference to FIG. 6, a simplified schematic is used to illustrate an EMP transfer between two electrical conductive metal pieces with a liquid medium containing a living microorganism biomass flowing between them in a method for harvesting biomass from an aqueous solution containing algae cells (single step extraction plus quantum fracturing). The cathode 3 requires a positive electrical connection point 5 - used for positive current input. Positive transfer polarizes the entire length and width of the cathode 3 and seeks a grounding source or anode 2, In order to complete an electrical circuit, the anode 2 requires a grounding connection point 4 which now allows an electrical transfer 6 to occur through a liquid medium containing a living biomass 1. The biomass 1 includes a liquid medium that contains a nutrient source mainly composed of a conductive mineral content and is used for sustaining life and reproduction of a living biomass 1. The liquid medium containing the nutrient source further allows positive electrical input to transfer between the cathodes 3 through the liquid medium/biomass 1 to the anode 2 and which only occurs when the liquid medium is present or flowing. Pulsing the electrical input phase contributes to cellular elongation 23 due to an electromagnetic field produced during an on cycle electrical phase. Any suitable number, duration and, for example, 60-80% duty cycle @ 1-2 kHz, of pulses can be used using the aforementioned watts. Elongation of the cell is due to a positive and negative polarity response due to conductive minerals consumed as part of their required nutrient uptake for growth and reproduction. Magnetic pulse response is useful in aiding in a further weakening process of the outer cellular wall structure prior to lysis completion. Once the pulsed electromagnetic field activates, the microorganism cells 23 magnetically align with the most responsive positive side facing the anode 2 and with the negative responsive side facing the cathode 3. During the off cycle electrical phase the cells are allowed to relax. At a high frequency rate of electrical input, the cells are repeatedly stretched and relaxed similar to a thin piece of metal being flexed back and forward until fracturing and breaking in two pieces occurs. This analogy is similar to the experience encounter by the biomass cells 23 during the on and off pulse phases which eventually aids in the lysis or fracturing process of the cell wall structure.

[0058] In reference to FIG. 7, a simplified schematic is used to illustrate a heat transfer example between the outer walls of the cathode 3 and/or anode 2 and into the liquid medium/biomass during the EMP process in a method for harvesting cellular mass and debris from an aqueous solution containing algae cells (single step extraction). An applied heating device 24 attaches to the outside wall surfaces of the cathode 3 and anode 2 which allows heat transfer to penetrate into the liquid medium containing a microorganism bio mass 1. Changes to the specific gravity of the liquid medium, which is mainly composed of water, by heating allows alteration in its compound structures which is mainly due to alterations to the hydrogen element which when altered, lessens the water density. This density change now allows a normally less dense material contained within a water column to sink or in this example, a lysed mass of cellular debris (cellular mass and debris),

[0059] In reference to FIG. 8, a simplified illustration is used to exhibit the difference between a normal sized biomass cell 25 in comparison to a cell 23 which has been exposed to an electrical charge. During the electrical on phase, pulsed electrical transfer 6 momentarily penetrates into intracellular components, which adsorb the energy transfer, resulting in momentary internal swelling to occur. This swelling produces pressure against the cell's wall structure 26 due to internal component swelling beyond allotted space allowances. During the off circuit phase, internal swelling decreases, however repeated on and off frequency creates an internal pumping action as the contained internal mass swells and is forced up against the cell wall structure. This repeated pressure combined with the electromagnetic field contributing to cellular pulsed elongation eventually causes external structural damage to the exterior wall with general damage resxilting in the form of lysis or fracturing. Once fracturing occurs, leakage of valuable internally substances from the cell structure and into the liquid medium occurs. [0060] In this embodiment, FIG. 9 illustrates a lower mounting location for a micron mixer 27 when in association with secondary tank 28 and containing a previously fractured biomass 29 suspended within a liquid medium. This liquid medium is then allowed to flow through a lower secondary tank outlet 30 where it is directed to flow through conduit 31 having a directional flow relationship with a liquid pump 32. Due to pumping action, the liquid is allowed a single pass through, or to re-circulate through the micron mixer via a micron mixer inlet opening 33. As liquid continues to flow through the micron mixer 27, microscopic bubbles 34 are produced which radiate outwards within the liquid column 35, forming a foam layer 36. As the process continues, the composed layer starts to rise upwards towards the surface of the liquid column 35. Once the foam layer 36 starts its upward journey towards the surface of the liquid column 35, the pump 32 is shut down, and thus the micronization process is complete. This allows all micron bubbles 34 produced at the lower exit point of the micron mixer 27 to rise to the surface and as they do, they start collecting valuable intracellular substances released into the liquid medium during the EMP process. This upward motion of the micron bubbles 34 also rubs or bumps into heavier downward -sinking cellular mass and debris, further allowing the release of trapped lighter valuable substances that have bonded with heavier- sinking cellular mass and debris remains. Once detached, these substances adhere to the micron bubbles 34 floating upwards towards the surface.

[0061 ] In reference to FIG. 10, a simple illustration is used to show a method for harvesting a foam layer 36 containing approximately ten percent of the original liquid medium mass/biomass 1 , As the foam layer 36 containing the valuable intracellular internal substances rises to the surface of the liquid medium 35, a skimming device 37 can be used to remove the foam layer 36 from the surface 38 of liquid medium 35. The skimming device 37 located at the surface area of the secondary tank 28 allows the foam layer 36 to be pushed over the side wall of the secondary tank 28 and into a harvesting container 39 where the foam layer 36 is allowed to accumulate for further substance harvesting procedures.

[0062] FIG. 1 1 illustrates one embodiment of a method and apparatus (system) as described herein for the harvest of useful substances from an algae biomass. Microorganism algae are grown in a containment system 40 and at the end of an appropriate growth cycle are transferred into the substance recovery process. The algae biomass are flowed through an optional micron bubble cavitation step 41, used to soften the outer cellular wall structure prior to other bio substance recovery processes.

[0063] After the cavitation step 41 an optional heat process 42 can be applied to change the gravity specifics of the liquid feed stock water containing the biomass. The heat option 42 allows a faster transfer of particular substances released during the harvest process. After the biomass has reached an appropriate heat range, it is then allowed to flow through an electromagnetic pulse field, the EMP station 43 where transiting biomass cells are exposed to the electromagnetic transfers resulting in the fracturing of the outer cellular wall structures. [0064] Once flowed through the EMP process 43, the fractured biomass transitions into a gravity clarifier tank 44 where heavier matter (ruptured cell debris/mass) 45 sinks down through the water column as the lighter matter (intracellular products) 46 rises to the surface where it allows an easier harvest. The heavier sinking mass 45 gathers at the bottom of the clarifier tank 44 where it can be easily harvested for other useful substances. After substance separation and recovery, the remainder of the water column 47 is sent through a water reclaiming process and after processing is returned back into the growth containment system 40. [0065] FIG, 12 illustrates another embodiment of a method and apparatus (system) as described herein for the harvest of useful substances from an algae biomass. Microorganism algae are grown in a containment system 48 and at the end of an appropriate growth cycle are then transferred into the substance recovery process. The substance recovery consists of the algae biomass being transfers into an optional heat process 49 where the biomass water column is subjected to heat prior to the EMP station 50. After the EMP process, the fractured biomass is then transferred over into a cavitation station 51 where micron bubbles are introduced at a low point in a water column containment tank 52. As the micro-bubbles rise through the water column, the valuable released bio substances (intracellular products) 53 attach to the rising bubbles which float to the surface of the water column allowing an easier and faster slamming process for substance recovery. After substance recovery, the remainder of the water column is sent through a water reclaiming process 54 and after processing is returned back into the growth system 48.

EXAMPLES

[0066] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.

Example 1 - Cell Lysing Method and Apparatus

[0067] In view of the interest in algae as a source of fuels and other materials, the development of methods and apparatuses for processing algal cells on a large scale is of great utility in processing the algal cells for such purposes. Such methods and apparatuses are described below.

[0068] One embodiment of a method for processing algal cells in suspension involves passing algal cells in aqueous suspension through a static mixer, where the static mixer creates cavitation effects, electrolyzing the suspension, and separating lysed cells from water in the suspension.

[0069] In particular embodiments, the method also involves entraining a pH or ORP modifying agent in the suspension, e.g., carbon dioxide. In such an embodiment, carbon dioxide typically is entrained in a static mixer. In a further refinement, a,. Becausesing alkaline materials may assist (make the process more efficient), agents may be used.

[0070] In certain embodiments, the method also involves collecting hydrogen gas generated by the electrolysis, e.g., at the mixer.

[0071] In certain advantageous embodiments, the suspension is a partial draw from an algal growth container, e.g., a draw taken 1, 2, or 3 times per day, or a draw taken once every 1, 2, 3,

4, 5, 6, or 7 days. Generally, the partial draw consists of approximately 10, 20, 30, 40, 50, 60,

70, 80, or 90 percent of the culture volume from an algal growth container or is in a range of 10 to 30. 30 to 50, 50 to 70, or 70 to 90 percent of the culture volume. Lysed and/or flocculated algal cells are separated from water in the suspension to provide recovered water, and the recovered water is sterilized and returned to the algal growth container.

[0072] In another embodiment, a system for processing algal cells in suspension includes a growth container in which algal cells are grown in suspension; a static mixer fluidly connected with the container through which at least part of the suspension is passed, thereby lysing at least some of said cells; and electrolysis electrodes in contact with the suspension, wherein an EMP is passed through the electrodes and through suspension between the electrodes.

[0073] In certain embodiments, the static mixer includes an injection port through which fluid may be entrained in the suspension; the static mixer also includes anode and cathode electrodes electrically connected to an electrical power source, e.g., as described herein.

[0074] In certain embodiments, the system also includes a biomass separator, a lipid extractor, and/or a hydrogen collector.

[0075] Some embodiments include a modified static mixer. Such a modified static mixer includes a body having a mixing throat through which liquid is passed, an injection port whereby fluid materials may be entrained in said liquid, and anode and cathode electrodes electrically separated from each other such that when a voltage is applied across said electrodes, an electrical current will pass through said liquid.

[0076] While such a mixer may be configured in many ways, in certain embodiments, one of the electrodes is within the body, and the other of the electrodes is located at the outlet in the body; one of the electrodes consists essentially of the body of the mixer, and the other of the electrodes consists essentially of an outlet ring insulated from the body.

7? [0077] Utilization of algae in methods for producing large quantities of algal oil or algal biomass has faced a number of hurdles, In addition to achieving efficient growth, those hurdles include efficiently separating algae biomass from culture fluid and lysing of cells to enable separation of oils or other products from cellular mass and debris. The problems are dramatically increased in large scale operations in contrast to laboratory scale processes. Indeed, many laboratory scale processes are not applicable to large scale operations due to physical limitations and/or cost limitations.

[0078] For example, in investigating these matters, no suggestion has been found for industrial scale application of EMP to the cell lysis of organisms of the taxonomy group: Archeaplastida and particularly Its sub group micro-algae. Indeed, conventional methods focus mainly on electrolysis of sludge (i.e., municipal and industrial waste) which is lower in pH and therefore has a higher or positive Oxygen Reduction Potential (ORP) or Mv reading. [0079] Electrocliemically, as pH lowers, there is a dramatic increase in the concentration of hydrogen ions and a decrease in negative hydroxyls or OH-ions (J. M. Chesworth, T. Stuchbury, J. R. Scaife, Introduction to Agricultural Biochemistry, pg 12. 2.2) Conversely, the higher the pH, the lower the ORI⁵. This correlation between high pH and negative Mv readings led to the conclusion that a resident charge on the cell wall can be transformed as energy to both facilitate cell lysing, but also to extract desired elements within the cell of benefit for the production of energy, pharmaceuticals and food products. From recent advances in X-ray crystallography biology of single cell organism, in this case cyanobaeteria or blue green algae, it was concluded that plant cell membranes are like the two ends of a battery, they are positive on the inside and negative on the outside, and they are charged up when solar energy excites electrons from hydrogen within the cell. The electrons travel up into the cell membrane via proteins that conduct them just like wires releasing the energy a plant needs to stay alive and from data on the accumulation of tetraphenylphosphonium within Chhrelϊa vulgaris cells, it can be estimated that these cells possess a membrane potential of - 120 to - 150 mV.

[0080] This negative potential is reflected in the observation of a vibrant cell colony's matrix pH level, where this measurement along with the correlate ORP (Mv reading) were taken to determine cell colony health. For example, a pH reading of 7 in an algae growth vessel correlate to an ORP reading of (+/-) +200Mv. When good cell health or log growth is attained; the pH of the matrix was noted to be pH 9.0; the corollary ORP reading was (+/-) -200Mv, Therefore, it can be surmised that the measure of a healthy algae cell colony can be determined by a negative Mv reading with each increase in one point of pH correlating to a decrease of roughly 200Mv. [0081 ] Most natural waters have pH values of between 5.0 and 8.5. As plants take in CO₂ for photosynthesis in aquatic ecosystems, pH values (and alkalinity) rise. Aquatic animals produce the opposite effect —as animals take in O₂ and give off CO₂, the pH (and acidity) is lowered. In steady state, the algae matrix reading was 7.0 pH and as hypertonic conditions are created through oxidation, the pH drops to below 7.0 and as low as 5.0 with an analogous ORP reading of +200 to +400Mv. When the cell wall does not collapse, but just becomes flaccid (as opposed to turgid); its contents are still encysted and the cell wall as represented in Donnan's law of equilibrhmi where the cell wall sets up an energy potential within its two opposite charged cell walls to survive until an isotonic state is regained. This is also referred to as the Gibbs-Donnan phenomenon. This is the state of equilibrium existing at a semipermeable membrane when it separates two solutions containing electrolytes, the ions of some of which are able to permeate the membrane and the others not; the distribution of the ions in the two solutions becomes complicated so that an electrical potential develops between the two sides of the membrane and the two solutions have different osmotic pressures. This charge is extremely balanced and is why cells can survive extreme adverse conditions only to rejuvenate when proper hypotonic conditions are present.

[0082] Live algal cells can be considered as an electrochemical fuel cell, where changing the polarity of the membrane from a live culture high pH and low ORP (150Mv) to a low pH and high ORP (+200Mv) results in the net gain of 350 Mv and an attendant release of hydrogen into the matrix, provided the electrical potential of the cell is broken and the cell wall is not just deflated. Such hydrogen production is one of the beneficial products obtainable from this invention.

[0083] By combining a number of approaches, it was discovered that a rapid, industrially scalable method of lysing and/or flocculating algal cells can be provided. Such methods can be applied in methods for obtaining useful products from algae, for example, extracting lipids, obtaining hydrogen gas, and/or obtaining algal cellular mass and debris, among others [0084] As a component to carry out such a process, the present methods can use a static mixer. Advantageous static mixers include but are not limited to those described in Uematsu et al., US Pat 6279611 , Mazzei, US Pat 6730214. Such mixers that assist in the generation of transient cavitation and/or mass transfer of gas to liquid can be used,

[0085] ft is surmised that by creating a rapid increase in ORP through manipulation or lowering the pH of the matrix, the electrical differential has the effect of abetting the electrolysis process in cell lysing with the attendant benefit of the generation of excess hydrogen as a byproduct of the cell wall content release.

[0086] Experimental work demonstrates that cell lysing was realized rapidly and economically with this combination. The theory of why a combination of cavitation, ultrasonics and pH modification works to lyse cells is empirical and the inventors are not intending to be bound by any particular explanation of the results.

[0087] The present process can advantageously include modification of ORP₅ usually through pH reduction. While such pH reduction (or other ORP modification) can be accomplished using a variety of acids and bases, it can also be accomplished using CO₂. Oxidation/reduction reactions involve an exchange of electrons between two atoms. The atom that loses an electron in the process is said to be "oxidized." The one that gains an electron is said to be "reduced." In picking up that extra electron, it loses the electrical energy that makes it "hungry" for more electrons. Chemicals like chlorine, bromine, and ozone are all oxidizers. [0088] ORP is typically measured by measuring electrical potential or voltage generated when a metal is placed in water in the presence of oxidizing and reducing agents. These voltages give us an indication of the ability of the oxidizers in the water to keep it free from contaminants. Thus, an ORP probe is really a millivolt meter, measuring the voltage across a circuit formed by a reference electrode constructed of silver wire (in effect, the negative pole of the circuit), and a measuring electrode constructed of a platinum band (the positive pole), with the fluid being measured in between. The reference electrode, usually made of silver, is surrounded by salt (electrolyte) solution that produces another tiny voltage. But the voltage produced by the reference electrode is constant and stable, so it forms a reference against which the voltage generated by the platinum measuring electrode and the oxidizers in the water may be compared. The difference in voltage between the two electrodes is measured.

[0089] Changing the pH of an aqueous solution can dramatically alter the ORP reading because of the effect of pH on the concentration of charged ions in the water. Thus, in the apparatuses and methods described herein, the pH and thus the ORP can be modified by contacting the water with one or more ORP or pH modifying agents. Advantageously, carbon dioxide gas can be used to lower the pH; bringing the pH down will raise the millivolt reading.

[0090] CO₂ can be entrained in the liquid medium in the form of micro or nanobubbles, e.g., entrained as micro or nanobubbles using a static mixer as described above. Entrainment of CO₂ gas in such a manner lowers the pH, modifying the ORP, which can lead to the production of additional hydrogen gas which can be collected.

[0091] In addition, entrainment of CO₂ (or other gas) as micro or nanobubbles can contribute to cell lysis as indicated below. Cavitation effects and/or ultrasonics can also be beneficially utilized to enhance cell lysis and/or cellular mass and debris flocculation. While such effects can be generated using an ultrasonic probe, they can also be generated using the cavitation effect of a static mixer with associated microbubble entrainment. Thus, passing the algac-containing medium through a static mixer with gas entrainment contributes to cell rupture and can assist cellular mass and debris flocculation.

[00921 As applied in the present system, EMP has the effect of lysing cells. However, an added benefit is the generation of hydrogen gas, which can be collected, e.g., for use as a fuel.

The quantity of hydrogen can be enhanced by ORP modification.

[0093] For some applications, it may also be beneficial to apply a magnetic field. For example, such a field can be applied in or adjacent to a static mixer. One way of accomplishing this is to locate strong magnets around the static mixer. In some cases, it may be beneficial to use alternating magnetic fields.

[0094] The present process can be configured to enhance the output of one or more of a number of different products. For example, products can be algal cellular mass and debris, lipids, selected proteins, carotenoids, and/or hydrogen gas.

[0095] In some applications, it may be desirable to generate cellular mass and debris using the methods and apparatuses described herein. Such cellular mass and debris can be produced in conjunction with enhanced or optimized production of one or more other products, or either without obtaining other products or without optimizing for obtaining other products.

[0096] Advantageously, the process can be configured to produce substantial amounts of hydrogen gas.

[0097] In a typical embodiment, it is desirable to obtain lipids from the algae, e.g., for use in biofuels and/or to provide algal omega~3 fatty acid containing oils (primarily eicosapentaenoic acid (20:5, n- 3; EPA) and docosahexaenoic acid (22:6, n- 3; DHA), For extracting such lipids, it is advantageous to lyse the cells, e.g., as described above. Release of lipids in such a manner allows a first separation to be carried out on the basis of different densities between the lipid- containing materia] and the bulk water. If desired, the lipids can be further extracted using other lipid extraction methods,

[0098] In some embodiments, this invention utilizes a plurality of the processes mentioned to produce enhanced cellular mass and debris separation, cell lysis, hydrogen production, and/or lipid separation. For example, electrolysis can be combined with ORP modification. [0099] Highly advantageously, a system is constructed to carry out the selected sub- processes as part of the overall algae processing method. One component useful in such a system utilizes a modified static mixer which has an anode and cathode built into the device. In use, the modified static mixer subjects the slurry to EMP, while concurrently injecting CO? gas or other ORP modifying agent through a venturi into the algae liquor as it flows through the device. The device can include a gas recovery system on either end for the recovery of gases (e.g., hydrogen) generated by the electrolysis process.

[00100] Such a modified static mixer is schematically illustrated in FlG. 13. Biomass slurry 1 is allowed entry into the mixer chamber via an intake pipe. Once inside the entry chamber the slurry 1 flows through an anode 2 and cathode 3 circuits which is powered by a direct current power supply 54. The anode and cathode electrodes, 2 and 3, only allow electrical transfers when a conductive liquid medium is flowed between them. In the case of this static mixer, the biomass slurry 1 is used to conduct the electrical transfer between the anode and cathode electrodes, 2 and 3. During electrical transfer, the biomass slurry 1 is further exposed to the transfer and with a partial amount of this transfer absorbed by the microorganism cells. Once electrical exposure occurs their cellular wall structures begin to weaken. After flowing through the anode and cathode circuit chamber, a non-conductive gasket 55 is used to isolate the two chambers apart as so to not allow and electrical transfer to the venturi chamber 56. The now structurally weaker cells can now be fractured by cellular / micron bubble collision caused by the venturi. To further increase efficiency of the substance separation process, a gas injection port 57 can be used to introduce chemical enhancements for substance fracturing and recovery. During cellular wall fracturing, a release of intercellular gases such as oxygen and hydrogen or others having value can be captured as part of the substance recovery system. These gases are directed to vent for capture at the end of the outlet 58 located at the static mixer exit port 59. Further exiting are the remains of the fractured biomass 29 which is also directed for recovery at the exit point 58. [00101] Thus, as indicated above, the system can advantageously be configured and used with partial draws from the growth container or reactor, e.g., a photo bioreactor. Also advantageously, the system can include and use a modified static mixer as described for extracting and flocculating (cellular mass and debris) from the matrix, capturing the generated hydrogen or excess oxygen, separating the cellular mass and debris from me water and returning the water back to the reactor, preferably after sterilization or filtration.

[00102] The method referred to herein as "Cascading Production", makes use of a percentage draw of (culture) liquor from the growth tank on a scheduled basis such as daily, every other day or weekly. The drawn (culture) liquor is then entrained through the electrolyzing mixing device and/or entrained through a mixer in conjunction with conventional electrolyzing method, such as an anode and cathode plate in the processing tank. Such processing can include ORP manipulation.

[00103] Viewed in a general sense, the methods and apparatuses described herein include a series of fluid manipulations along a process flow with the specific goal of extracting valuable by-products contained in algal cells. As described briefly above, as the algae is grown in tanks, e.g., salt water tanks, of diverse configurations such as outdoor growth ponds, open tanks, covered tanks, or photo bioreactors (PBR), a portion of the solution or liquor is drawn on a scheduled basis. This draw schedule is determined but not limited to the following observations taken on a daily basis of density, pH and/or ORP. For example, it has been noted that the pH of an outdoor pond is higher in the evening than during the morning, due to CO₂ absorption and the process referred to as respiration which occurs at night. The difference can be as high as 3 pH points or 600Mv. Therefore, one would draw a significant portion of the growth pond in the evening as the pH is now at 8.5-10 (early morning readings would compare at (6.-7). In a reactor or PBR, the same principle applies, but in this case one observes the log stages of growth and draws up to 75% of the growth fluid (matrix) when the pH reaches 8.5-9. All these indicators use conventional measuring equipment incorporated into a plant process computer controller, that would control the SSE process and signal when it is time to harvest. To determine when it is time to harvest, several indicators in the growth vessel, such as PH, ORP, Mv, salinity, size of cells, etc., can be evaluated. [00104] The remaining percentage of undrawn fluid is kept as an incubator for the recycled water and used to start a new log phase of algae growth. The drawn liquor (also referred to herein as "culture"),

[00105] Microorganism algae are grown in a containment system and at the end of an appropriate growth cycle are transferred into the substance recovery process. The algae biomass are flowed through an optional micron bubble cavitation step, used to soften the outer cellular wall structure prior to other bio substance recovery processes.

[00106] After the cavitation step an optional heat process can be applied to change the gravity specifies of the liquid feed stock water containing the biomass. The heat option allows a faster transfer of particular substances released during the harvest process. After the biomass has reached an appropriate heat range, it is then allowed to flow through an electromagnetic pulse field, the EMP station where transiting biomass cells are exposed to the electromagnetic transfers resulting in the fracturing of the outer cellular wall structures.

[00107] Once flowed through the EMP process, the fractured biomass transitions into a gravity clarifier tank where heavier matter (cellular mass and debris) sinks down through the water column as the lighter matter rises to the surface where it allows an easier harvest, The heavier sinking material {cellular mass and debris) gathers at the bottom of the clarifier tank where it can be easily harvested for other useful substances. After substance separation, and recovery, the remainder of the water column is sent through a water reclaiming process and after processing is returned back into the growth system.

[00108] During this period of "cracking", the static mixer can inject one or more ORP modifiers, which can be or include pH modifiers such as CO₂. While CO₂ is preferred, alternative or additional pH or ORP modifiers can be used which accomplish the basic function of altering the pH value and its corollary ORP value as represented in Mv. Any suitable static mixer can be used; the methods, systems and apparatuses described herein are not limited to any particular type of mixer or the associated electrolyzing method. Such a mixer can incorporate a cathode and anode connected to a voltage regulator, which preferably flips polarities so as to reduce scaling on the probes. The anode and cathode are powered by a DC energy source, such as a battery, generator, transformer or combination thereof. The DC voltage can be set to variable outputs to adjust to algae mass in the cracking tank. [00109] As the fluid is entrained through the Venturi mixer, it is therefore admixed with CO₂, subjected to EMP field as mentioned above, and through the continuous mixing, a plurality of micron bubbles are generated, creating a cavitated, or slurry of micron bubbles of both CO₂ and alga mass. A combination of CO₂ entrainment, electrolysis, and mixing can be empirically selected, e.g., based on the desired separation of products from the algae cells and/or flocculation of the mass to the surface of the water.

[00110] For example, in a recent test, CO₂ was applied to attain a drop from pH 8.5 to 6.5 with a corresponding increase from -200Mv to + 250Mv and the fluid was electrolyzed using a DC 6 Volts power supply and complete flocculation and cell iysing (as examined under a microscope) was obtained within a period of 20 minutes. However, this combination and these parameters are only exemplary, and can be examined to determine optimum values. Desired results can be farther correlated with processing variables, e.g., to establish protocols based on pH values, ORP reading, cell density and alga species. Upstream PH modification, prior to SSE, may help the SSE process,

[00111] When electrolysis is utilized, concurrent with the process of cracking (Iysing) hydrogen gas (H+) is released at the cathode. This hydrogen can be safely recovered and trapped in a tank through a hydrogen recovery valve , placed on the cathode end of an electrolyzing unit or at the end of the static mixer. If one alters the pH values by using a base chemical compound, e.g., potassium hydroxide, sodium hydroxide, calcium hydroxide or magnesium hydroxide, one would now create an excess of free oxygen at the anode probe. In this instance, one would draw as above a certain portion of algae mass at a pH value of 8.5 and raise that value to approximately pH 11 or roughly -250Mv to -700 Mv and create a matrix high in negative hydroxyls or -OH. The dissociation of the free oxygen would then be created as the matrix returned to 7,0 upon cell cracking. In this case, one would incorporate a safe recovery system for this oxygen.

[001 12] In this system, once the cellular mass and debris is cracked, depending on the conditions, it may flocculate to the surface of the water or may sink. The cellular mass and debris is generally a composite of broken cell wall, lipid, carbohydrate and chlorophyll (A). In many cases, within a few hours, floe at the surface sinks to the bottom of the tank. While some of the lipid may remain on the surface, a significant fraction of the lipid (which may be most of the lipid) is still associated with chlorophyll and/or other cellular components and will sink with the rest of the cellular mass and debris.

[001 13] The remainder of the water is now of about 7.0 pH, with a high CO₂ concentration. (only if pH was adjusted, otherwise the PH will be that of the inbound slurry) This water (slurry is processed) and its cracked biomass (cellular mass and debris) is now entrained or flowed to a water sterilizing tank after passing through a filtration unit, where a number of systems can be used to separate out the organic mass from the water. These systems can, for example, be plane separators, filters, vortex separators or any other method that performs the function of delivering a separated mass. The separated cellular mass and debris is drawn to a cellular mass and debris collection vessel and the water is sent on for sterilization in tank. After sterilization, the recovered water can be used to replenish tank.

[001 14] In one embodiment, the system includes a modified Venturi mixer nozzle, e.g., as illustrated in FIG. 13. As previously indicated, the slurry input pipe is insulated in the middle, or anywhere else along the length of pipe with a large rubber gasket or other electrically insulating material so as to separate the polarity of the anode and cathode. The two ends of the tube can be electrified from source DC input or include probes within the tubes that have the purpose of conducting electricity. The modified Venturi introdiices CO₂ gas or other admixture with the purpose of altering pH and ORP through an inlet tube into a low pressure zone designed within the geometry of the tube; according to Bernoulli's principle. At the exit of the venturi tube, a device can be installed for the purpose of capturing the hydrogen created during the EMP process. One can add obstructions within the venturi tube to impact the fluids flow to increase turbulence and create a plurality of micron-bubbles.

Example 2 - Quantification of Lipid Extraction and Identification of Optimal EMP Extraction

Parameters

[001 15] In the experiments described below, quantification of lipid extraction using an EMP apparatus as described herein and identification of optimal extraction parameters are described.

The results described below correspond to the data in FIG, 14.

Test 1 ;

[00116] In order to quantify lipid extraction from an EMP unit as described herein, the following experiment was performed. A batch of NcmnocMoropsis oculata was processed through the 6-inch EMP unit to extract the lipids. The batch was gravity fed through the EMP unit at a flow rate of about IL/min. A total of 20.8 L of algae culture was processed. The processed stream was scooped off the top layer after collection for lipid analysis.

Control Batch Details:

Dry mass concentration: 433 mg/L

Lipid content: 5.5% of dry mass (23.86 mg/L) pH: 7.1

Conductivity: 8.82 mS/cm

Extraction Process Details:

Extraction sample volume: 20.8 L

Flow rate: 1 L/rnin

Voltage: 4.3 V

Electric current: 22 Amp

[001 17] Results: The extraction sample was analyzed by the Folch method. The extracted lipid weighed 0. 4481 g. The amount of lipid originally present in the 20.8 L of algae batch before processing was 0. 4965 g. This corresponds to an extraction efficiency of 90.2% through the EMP unit.

Test 2:

[00118] In order to quantify lipid extraction from an EMP unit as described herein, the following experiment was performed. A batch of Nannochloropsis ocidata was processed through the 6-inch EMP unit to extract the lipids, The batch was gravity fed through the EMP unit at a flow rate of about 1 L/min. A total of 9.2 L of algae culture was processed. The processed stream was collected in a lipid collection apparatus that was designed to have tapered long neck to collect the lipid layer floating at the top.

Control Batch Details:

Dry mass concentration: 207 mg/L

Lipid content: 13% of dry mass (26.91 mg/L) pH: 6.8

Conductivity: 9.31 mS/cm

Extraction Process Details: Extraction sample volume: 9.2 L Flow rate: 1 L/min Voltage: 3.4 V Electric current: 20 Amp

[00119] Results: The extraction sample was analyzed by the Folch method. The extracted lipid weighed 0.2184 g. The amount of lipid originally present in the 9.2 L of algae batch before processing was 0.2477 g. This corresponds to an extraction efficiency of 88.2% through the EMP unit.

Test 3

[00! 20] In order to quantify lipid extraction from an EMP unit as described herein, the following experiment was performed. A batch of Nannochϊoropsis oculata was processed through the 6-inch EMP unit to extract the lipids. The batch was gravity fed through the EMP unit at a flow rate of about 1 L/min. A total of 1 1 L of algae culture was processed. The processed stream was scooped off the top layer after collection for lipid analysis.

Control Batch Details:

Dry mass concentration: 207 rng/L

Lipid content: 13% of dry mass (26.91 mg/L) pH: 6.8

Conductivity: 9.31 mS/cm

Extraction Process Details: Extraction sample volume: 1 1 L Flow rate: 1 L/min Voltage: 3.4 V Electric current: 20 Amp

[00121] Results: The extraction efficiency was 95.25% through the 6-inch EMP unit for the tested algae batch.

Test 4

[00122] In order to quantify lipid extraction from an EMP unit as described herein, the following experiment was performed. A batch of Nannochloropsis oculata was processed through the 6-inch EMP unit to extract the lipids. The batch flow rate was regulated using a flowmeter and a pump, 2 liters of algae culture was processed. The processed stream was collected in a 2 liter volumetric flask, and the top lipid layer was recovered for analysis. Control Batch Details:

Dry mass concentration: 410 mg/L

Lipid content: 8.2%of dry mass (33.62 mg/L) pH: 7.1

Conductivity: 8.99 mS/cm

Extraction Process Details: Extraction sample volume: 2.01 L Flow rate: 1.5 L/min Voltage: 12.4 V Electric current: 18 Amp

[00123] Results: The extraction efficiency was 90.7% through the 6-inch EMP unit for Ae tested algae batch.

Test S

[00124] In order to quantify lipid extraction from an EMP unit as described herein, the following experiment was performed. A batch of Nannochloropsis oculata was processed through the 12-inch EMP unit to extract the lipids. The batch flow rate was regulated using a flowmeter and a pump. 1.87 liters of algae culture was processed. The processed stream was collected in a 2 liter volumetric flask, and the top lipid layer was recovered for analysis.

Control Batch Details:

Dry mass concentration: 800 mg/L

Lipid content: 19.9%of dry mass (159.2 mg/L) pH: 7.6

Conductivity: 8.15 mS/cm

Extraction Process Details: Extraction sample volume: 1.87 L Flow rate: 0.2 gal/min (0.756 L/min) Voltage: 4.8 V Electric current: 20.2 Amp

[00125] Results; The extraction efficiency was 12.2% through the 12-inch EMP unit for the tested algae batch.

Test 6: [00126] In order to quantify lipid extraction from an EMP unit as described herein, the following experiment was performed, A batch of Namiochloropsis ocxύata was processed through the 12-inch EMP unit to extract the lipids. The batch flow rate was regulated using a flowmeter and a pump, 1.87 liters of algae culture was processed. The processed stream was collected in a 2 liter volumetric flask, and the top lipid layer was recovered for analysis.

Control Batch Details:

Dry mass concentration: 500 mg/L

Lipid content; 16,l5%of dry mass (80.75 mg/L) pH; 7.5

Conductivity: S.18 mS/cm

Extraction Process Details: Extraction sample volume: 1.87 L Flow rate: 1.13 L/min Voltage: 4.7 V Electric current: 20 Amp

[00127] Results: The extraction efficiency was 51.5% through the 12-inch EMP unit for the tested algae batch.

Test 7:

[00128] In order to identify the optimal EMP extraction parameters for a given algae batch, the EMP was tested in a matrix of wide range of parameters. A batch of Nannochloropsis oculata was processed through the 6-inch EMP unit to extract the lipids. The batch flow rate was regulated using a flowmeter and a pump. Individual samples that comprised the matrix of testing were collected in small 116 ml bottles. The cellular mass and debris at the bottom and the water were syringed out leaving only the top lipid layer in the extraction sample bottle.

Control Batch Details:

Dry mass concentration: 210 mg/L

Lipid content: 24% of dry mass (50 mg/L) pH: 7.8

Conductivity: 7.89 mS/cm

Extraction Results:

Extraction sample volume: 116 ml

The amount of lipid originally present in the 1 16 ml algae sample before processing; 5.8 mg The extraction sample was analyzed by the Folch method. The relevant parameters comprising the matrix of testing conditions and the extraction efficiency are tabulated in Table 1.

Table 1 , Extraction efficiency at different flow rates and current strengths

[00129] Inference: The most optimal conditions for lipid extraction for this batch of algae look to be 0.25 gal/min and 15 Amp. The efficiency decreases gradually around this set of conditions in the tested matrix, At higher currents at 0.25 gal/min, the energy input is probably too high to the detriment of algae causing them to destmct, At lower currents at 0.25 gal/min, and at lower flow rates, the energy input is too less to fully extract the lipids from algae.

Test s

[00130] In order to quantify lipid extraction from an EMP unit as described herein, the following experiment was performed. A batch of Nannochloropsis oculata was processed through the 6-inch EMP unit to extract the lipids. The batch flow rate was regulated using a flowmeter and a pump. Samples were collected either in 1 16 ml bottles or 400 ml bottles. The cellular mass and debris at the bottom and the water were syringed out leaving only the top lipid layer in the extraction sample bottles.

Control Batch Details:

Dry mass concentration: 320 mg/L

Lipid content: 18% of dry mass (57.6 mg/L) pH: 7.3

Conductivity: 7.93 mS/cm

Extraction Process Details: Flow rate: 0.95 L/min Voltage: 5.3 V Current: 20 A

Results:

Extraction sample 1 :

Volume: 412 ml

Extraction efficiency: 83.31%

Extraction sample 2:

Volume: 1 16 ml

Extraction efficiency: 80.69%

Extraction sample 3:

Volume: 116 ml

Extraction efficiency: 95.64%

Test 9:

[00131] In order to identify the optimal EMP extraction parameters for a given algae batch, the EMP apparatus as described herein was tested in four different sets of conditions. 20 liters of a Nannochloropsis oculata batch from the grow room was processed through the 6-inch EMP unit. The batch flow rate was regulated using a flowmeter and a pump.

Control Sample Details (Sample # 1130-0): Dry mass concentration: 320 mg/L Lipid content: 11% of dry mass (35 mg/L) pH: 7.5 Conductivity: 8.15 mS/cm

[00132] The algae batch was processed under various flow rate and energy input conditions as listed below: Sample 1130-3: Flow rate = 0.25 gal/min, Voltage = 3.7 V, Current = 15 Amp Sample 1130-4: Flow rate = 0.25 gal/min, Voltage = 4.0 V, Current = 20 Amp Sample 1 130-8,9: Flow rate = 0.38 gal/min, Voltage = 4.0 V, Current = 20 Amp Sample 1130-12: Flow rate - 0.38 gal/min, Voltage = 3.7 V₅ Current = 15 Amp

[00133] Samples were collected in 400 ml bottles. The cellular mass and debris at the bottom and the water were syringed out leaving only the top lipid layer in the extraction sample bottles. The samples were analyzed by CSU LB-IlRMES using the Folch Method.

[00134J Results: The most optimal conditions for lipid extraction for this batch of algae look to be 0.38 gal/min; 3.7 V; 15 Amp.

Table 2

Test 10:

[00135] The new Pipe EMP equipment along with MX cavitation and heat was tested and compared with previous tests. A batch of Nannochioropsls oculaia was processed through the Pipe SSE system. The components of the Pipe SSE system are the pipe EMP unit, a heat strip system around the pipe EMP unit, and an MX cavitation unit. The MX cavitation unit precedes the pipe EMP unit. The MX cavitation unit and the heating system around the EMP unit could be used optionally. The cavitation was done for 1 minute. The batch flow rate was regulated using a flowmeter and a pump. Samples were collected in 120 ml bottles. The cellular mass and debris at the bottom and the water were syringed out leaving only the top lipid layer in the extraction sample bottles.

Control Batch Details:

Dry mass concentration: 280 mg/L

Lipid content; 21% of dry mass pH: 7.7

Conductivity: 7.42 mS/cm Extraction Results and Observations: Extraction sample volume: 120 ml

Table 3 - Extraction results and observations of the Pipe SSE testing that included both MX cavitation and heatin

cellular mass and debris received less heat than that at 0.25 gal/min

[00136] The following table (Table 4) shows the extraction results and observations of the Pipe EMP testing that included only of MX cavitation and heating or neither. This can be used for comparison with the similar testing conditions in the table above.

Table 4 - Extraction Results

[00137] It looked like heat resulted in enhanced electrolysis that resulted in the cellular mass and debris to flocculate better. When the heat was high (as in @ 0.25 gal/min), all the flocculated cellular mass and debris sunk leaving a clear thin lipid layer at the top. The sinking was probably because the density of heated water is markedly lower than that of cellular mass and debris. When the heat is low (as in @ 0.50 gal/min), all the flocculated cellular mass and debris remained at the top stuck to the lipid. This is probably because the differential densities of water and cellular mass and debris is not big enough to cause instant sinking of cellular mass and debris, but the applied heat was still enough to flocculate the cellular mass and debris. Either way, it was seen that when there was heat the cellular mass and debris flocculated either at the top or at the bottom, but when there was no heat they remained suspended as seen normally with the previous 6-inch and 12-inch EMP units without heat.

[00138] Another strong possibility is that when the cellular mass and debris flocculates and sinks to the bottom with the application of heat, some of the extracted lipid that was stuck to the cellular mass and debris could be carried along with the cellular mass and debris to the bottom. As a result, the extraction efficiency as analyzed from the lipid at the top clear layer could be lower. Conversely, when the cellular mass and debris flocculated and floated at the top, even if all of the lipids inside the algae cells may not have been extracted, the non-extracted lipids may still remain at the top along with the extracted lipids.

[00139] Another observation was the effect of current in sinking the cellular mass and debris when heat was applied. In the first table, in the column corresponding to 0.25 gai/min, the speed at which the cellular mass and debris sank was directly proportional to the amount of electric current supplied. Even at the flow rate 0.50 gal/min, where all the cellular mass and debris floated because of lower heal, the cellular mass and debris corresponding to the sample with 20 Amperes of electric current sank after 1 day, whereas the cellular mass and debris corresponding to the samples with lower current continued to float after 1 day.

Test 1 1 :

[00140] In order to obtain lipid extraction at the highest efficiency possible for a given batch of algae, an EMP apparatus as described herein was tested in different sets of conditions. A batch of Nannochloropsis oculata was processed through the 6-inch EMP unit to extract the lipids. The batch flow rate was regulated using a flowmeter and a pump. Samples were collected in 1 liter bottles. The cellular mass and debris at the bottom and the water were syringed out leaving only the top lipid layer in the extraction sample bottles.

Control Sample Details (Sample # 20100104-10):

Dry mass concentration: 285 mg/L

Lipid content: 6.67% of dry mass (19 mg/L) pH: 8.4

Conductivity: 7.99 mS/cm

Extraction Results:

Extraction sample volume: 1 L

The amount of lipid originally present in the 1 L algae sample before processing: 19 mg

[00141] The samples were analyzed by CSULB -IIRMES using the Folch Method. The relevant parameters of different testing conditions and the extraction efficiencies are tabulated in following table.

Table 5 - Parameters of Testing Conditions and Extraction Efficiencies

Voltage: 3.7 V Voltage: 3,7 V

Extraction efficiency; 39% Extraction efficient

Sample # 20100104-14

Current; 20 Amp

Voltage; 4.0 V

Extraction efficiency: 41%

Sample # 20100104-15

Current: 19 Amp

Voltage: 3.9 V

Extraction efficiency; 98%

[00142] The highest extraction efficiencies 98% and 96% were obtained at 0.25 gal/min; 19 Amp; 3.9 V and at 0.50 gal/min; 18 Amp; 3.8 V for the tested algae batch.

Tests 12 and 13:

[00143] The effect of overnight storing in darkness and cold on lipid extraction efficiency was examined. Samples from the same algae batch were tested in Test 12 and were tested on the following day in Test 13. The same algae batch tested in Test 12 was tested on the following day (the same tests were run on the same original algae culture; one test occurred on the day the live sample was drawn from the growth tank, i.e., real-time, and the 2ⁿ day the remainder of the sample was tested after it rested overnight). A batch of Nannochloropsis ociύata was processed through the Pipe SSE system. The components of the Pipe SSE system are the pipe EMP unit, a heat strip system around the pipe EMP unit, and an MX cavitation unit. The MX cavitation unit precedes the pipe EMP unit. The MX cavitation unit and the heating system around the EMP unit could be used optionally. The cavitation was done for 1 minute. The batch flow rate was regulated using a flowmeter and a pump. Samples were collected in 120 ml bottles. The cellular mass and debris at the bottom and the water were syringed out leaving only the top lipid layer in the extraction sample bottles.

Table 6 - The control sample details ertaining to the first day and the second da after stora e.

Extraction Results; Extraction sample volume: 120 ml

Table 7 - Relevant parameters of the testing conditions and the extraction efficiencies

[00144] The extraction efficiencies are in general lower than the earlier Pipe SSE experiments. This is probably because the extraction samples were left to sit too long before recovering the top lipid layer. Usually there is some cellular mass and debris that is found in the top lipid layer, but all of it had sunk as a result of letting the samples sit for too long, and along with it some of the lipid could have sunk as well. Comparing the extraction efficiencies observed on the first day and the second day, there does not seem to be any improvement in extraction due to the overnight storage in darkness and cold.

Example 3 - Use of Cavitation and EMP to Harvest Carbohydrates and Proteins [00145] FIG. 14 shows results from a test procedure for harvesting carbohydrates and proteins from algae. The test procedure was performed as follows. The algae slurry was first processed through the EMP unit at room temperature. The EMP processed slurry was collected in a storage tank. It was then cavitated through the MX unit. The cavitated slurry was then allowed to sit for a few minutes. A thick mass of algae cellular mass and debris raised to the top and remained floated. The floating cellular mass and debris was collected off the top for analysis. [00146] The algae samples collected through the Inverse SSE process was analyzed by Anresco Laboratories, San Francisco. The samples were analyzed for lipid, protein and carbohydrate content of the algae. The analysis by Anresco Laboratories gave the total mass of protein, lipid or carbohydrate in a given sample (say 'x' mg).

[00147] The dry mass concentration of the algae batch processed (say 'dl' mg/L) was measured before the Inverse SSE process, The volume of the algae batch collected in the storage tank from where the final floating cellular mass and debris was collected off the top was also known (say 'V L). The dry mass concentration of the remnant solution after the collection of floating cellular mass and debris off the top was also measured (say ^*d2^* mg/L). From these the mass of algae cellular mass and debris (say 'M' mg) collected off the lop of the storage tank was calculated as follows:

M = (dl - d2) x V [00148] Then, the individual composition of protein, for example, was calculated as follows:

Protein composition = x/M mg of prαtein/mg of algae dry mass.

[00149] For this experiment, three small samples were taken from the sample jar (it was observed that the algae collected off the top from the process was sticky, agglomerated and floating on water). Based on the dry mass measurements and the volume of algae slurry processed, the amount of biomass collected off the top through the Inverse SSE process was 600 mg. The protein quantity alone as analyzed by Anresco Laboratories amounts to 1400 mg. As the amount of protein should not be higher than the amount of biomass, the amounts measured could be due to increased protein numbers that resulted from sampling methods, e.g., there might have been more algae in the three drawn samples than there might be if they were uniformly mixed. Nonetheless, these results demonstrate that the apparatuses and methods described herein can be used to harvest protein as well as fat from algae cells (see Table 8 below).

Table 8 - Results from three samples of Algae marked 0413:1-3

Other Embodiments

[00150] One skilled in the art would readily appreciate that the present invention is well adapted to obtain the ends and advantages mentioned, as well as those inherent therein. The methods, systems, and apparatuses described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention and are defined by the scope of the claims, [00151] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. For example, variations can be made to the configuration of the tanks, materials utilized, ORP modifying agents, and algal species grown. Thus, such additional embodiments are within the scope of the present invention and the following claims. [00152] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions, any equivalents of the features shown and described or portions thereof are excluded, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. [00153] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[00154] Also, unless indicated to the contrary, where various numerical values or value range endpoints are provided for embodiments, additional embodiments are described by taking any two different values as the endpoints of a range or by talcing two different range endpoints from specified ranges as the endpoints of an additional range. Such ranges are also within the scope of the described invention. Further, specification of a numerical range including values greater than one includes specific description of each integer value within that range.

[00155] Thus, additional embodiments are within the scope of the invention and within the following claims.

Claims

CLAIMS:

1. An apparatus for harvesting at least one intracellular component from algae cells in aqueous suspension, the apparatus comprising: a) at least one ørst electrical conductor that acts as a cathode and a second electrically conductive housing that acts as an anode, the at least one first conductor being disposed within the housing, such that a space is defined between the exterior of the first conductor and an interior of the housing, providing a flow path for the aqueous suspension, wherein at least a portion of one or both surfaces of the first conductor and the housing has been removed to create at least two spiral grooves separated by at least one land that reduces or prevents algae cell buildup on or around the first conductor and the housing; b) an electrical power source operably connected to the first conductor and the housing for providing a pulsed electrical current that is applied between the first conductor and the housing and the aqueous suspension for rupturing the algae cells resulting in a mass of ruptured algae cells and debris and release of intracellular components from the algae cells in the aqueous suspension; and c) a secondary tank that is operably connected to the first electrical conductor and the housing such that the aqueous suspension can flow from the flow path into the secondary tank for separation of the at least one intracellular component from the ruptured algae cells in aqueous suspension.

2. The apparatus of Claim 1 , wherein the first conductor is a metal tube.

3. The apparatus of Claim 1, wherein the first conductor and second housing are each metal tubes.

4. The apparatus of Claim 3, wherein the first conductor and second housing are metal tubes of circular shape.

5. The apparatus of Claim 3, wherein the metal tubes are of different shapes.

6. The apparatus of Claim 4, wherein the inner diameter of the metal housing and the outer diameter of the first conductor differ in size on the order of 0.050 inch.

7. The apparatus of Claim 1. wherein the housing is a metal tube and the at least one electrical conductor comprises a plurality of spaced apart electrical conductors, the electrical conductors being separated from each other by electrically insulating elements; and a multiplicity of flow paths being created between the housing and each of the plurality of spaced apart electrical conductors.

8. The apparatus of claim 7, wherein each of the plurality of electrical conductors are metal tubes.

9. A method of harvesting at least one intracellular component from algae cells in aqueous suspension comprising the steps of: a) providing the apparatus of claim 1 , the apparatus further comprising an aqueous suspension comprising conductive minerals and algae cells wherein the aqueous suspension is disposed in the flow path of the apparatus; b) applying a sufficient amount of a pulsed electrical current to the at least one first conductor and the housing and aqueous suspension for caused alternature expansion and contraction of the cell contents thereby rupturing the algae cells resulting in a mass of ruptured algae cells and debris and release of intracellular components from the algae cells in the aqueous suspension; c) flowing the aqueous suspension containing the mass of ruptured algae cells and debris and released intracellular components to the secondary tank for separating the intracellular components from the biomass and aqueous suspension; and d) separating the at least one intracellular component from the mass of ruptured algae cells and debris and aqueous suspension.

10. A method of harvesting a mass of ruptured algae cells and debris from an aqueous suspension comprising algae cells, the method comprising the steps of: a) providing the apparatus of claim 1 , the apparatus further comprising an element disposed in the secondary tank for producing microbubbles, an aqueous suspension comprising conductive minerals and algae cells wherein the aqueous suspension is disposed in the flow path of the apparatus, and a pump disposed in the secondary tank for circulating the aqueous suspension; b) applying a sufficient amount of a pulsed electrical current to the at least one first conductor and the housing and aqueous suspension for rapturing the algae cells resulting in release of intracellular components from the raptured algae cells and a mass of raptured algae cells and debris in the aqueous suspension; c) flowing the aqueous suspension containing the released intracellular components and mass of ruptured algae cells and debris to the secondary tank for separating the biomass from the released intracellular components and the aqueous suspension; d) activating the pump and the element for producing microbubbles resulting in a plurality of microbubbles that attach to the released intracellular components and float upwards in the aqueous suspension and the sinking of the mass of ruptured algae cells and debris doλvnwards in the aqueous suspension; and e) separating the mass of ruptured algae cells and debris from the released intracellular components and aqueous suspension.

11 , The method of claim 10. wherein the element disposed in the secondary tank for producing microbubbles is a mixer.