FROM AQUEOUS STREAMS TO PRODUCE PRODUCTS USING A HOLLOW-FIBER MEMBRANE

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of oil separation from aqueous streams, and more particularly, to systems and methods for making and separating oils from various processing streams, and using the recovered oil, chemicals, purified water, biomass or other products for various downstream uses, including potentially further processing one or all of the products further.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is described in connection with the recovery of commercially valuable oils, chemicals, de-oiled water, biomass or other products from aqueous streams and other sources.

United States Patent No. 7,297,261, issued to Bomberger, et al., relates to a system and method of using a solvent for the removal of lipids from fluids such as blood. Briefly, this patent teaches systems and methods for removing lipids from a fluid (such as blood plasma, or from lipid-containing organisms), wherein a fluid is combined with at least one extraction solvent, which causes the lipids to separate from the fluid or from lipid-containing organisms. The separated lipids are then removed from the fluid. The extraction solvent is removed from the fluid or at least reduced to an acceptable concentration enabling the delipidated fluid to be administered to a patient without the patient experiencing undesirable consequences. The processed fluid may be administered to a patient who donated the fluid, to a different patient, or stored for later use.

United States Patent No. 7,868,195, issued to Fleischer, et al., relates to a system and method for extracting lipids from and dehydrating wet algal biomass. Briefly, this patent teaches methods, including centrifugation, increasing the solid content of a wet algal biomass to between approximately 10% and 40%, mixing the centrifuged algal biomass with an amphiphilic solvent, heating the mixture and separating the amphiphilic solvent from the algal biomass. The resulting amphiphilic solvent, water and lipids, are further processed by evaporating the amphiphilic solvent from the water and the lipids and separating the water from the lipids. Examples of amphiphilic solvents include acetone, methanol, ethanol, isopropanol, butanone, dimethyl ether, and propionaldehyde. The method can also include filtering the wet algal biomass through a membrane to increase a solid content of the wet algal biomass to between approximately 10% and 40%.

U.S. Patent No. 7,431,952, issued to Bijl and Schaap, discloses extraction of a microbial or single cell oil, for example comprising one or more polyunsaturated fatty acids (PUFAs), directly from microbial cells to avoid the need for solvents. After fermentation, the microbial cells are pasteurized, washed and the cell walls lysed or disrupted by a mechanical (e.g. homogenization), physical (boiling or drying), chemical (solvents) or enzymatic (cell wall degrading enzymes) technique. The oil (containing the PUFA) is then separated from the resulting cell wall debris. This is achieved by centrifugation, which results in an oily phase (top layer) that contains the oil, which can be separated from an aqueous phase (containing the cell wall debris). The oil can then be extracted and if necessary the PUFA can be purified or isolated from the oil.

U.S. Patent No. 6,166,231, issued to Hoeksema, relates to a method of separating edible oil from biological material. A biomass slurry containing microbial material in an aqueous suspension is collected. The slurry is typically placed in a centrifuge and then in a homogenizer. The resulting slurry is fed into a contacting device, such as a packed column, and mixed with a solvent that is essentially immiscible in water, for example hexane. The solvent extracts the oil from the biomass slurry and then separates from the slurry. Edible oil is recovered from the solvent and further processed.

United States Patent Application Publication No. 2011/0086386, filed by Czartoski, et al., relates to algae biomass fractionation. Briefly, this application is said to teach a method of fractionating biomass, by permeability conditioning a biomass suspended in a pH adjusted solution of at least one water-based polar solvent to form a conditioned biomass, contacting the pH adjusted solution with at least one non- polar solvent, partitioning to obtain a non-polar solvent solution and a polar biomass solution, and recovering cell and cell derived products from the non-polar solvent solution and polar biomass solution. The invention is also said to include products recovered from the above method. Finally, a method of operating a renewable and sustainable plant for growing and processing algae is also said to be taught.

United States Patent Application Publication No. 2010/0233761, filed by Czartoski, et al., relates to algae biomass fractionation. Briefly, these applicants are said to teach a method of fractionating biomass, by permeability conditioning the biomass suspended in a pH adjusted solution in at least one water-based polar solvent to form a conditioned biomass, contacting the pH adjusted solution with at least one non- polar solvent, partitioning the input to obtain an non-polar solvent solution and a polar biomass solution, and recovering cell and cell derived products from the non-polar solvent solution and polar biomass solution.

U.S. Patent Application Publication No. 2009/0053342 filed by Streekstra and Brocken, provides a process for the production of a microbial oil comprising culturing a micro-organism in a two stage fermentation process where, in a last stage that precedes the end of fermentation, the carbon source is: consumed by the micro-organisms at a rate greater than it is added to the medium; added at a rate 0.30 M carbon/kg medium; or is rate limiting on the growth of the micro-organism. The micro-organisms thus have the carbon source restricted so that they preferentially metabolize fats or lipids other than arachidonic acid (ARA), so increasing the proportion of ARA in the cells.

SUMMARY OF THE INVENTION

The present invention is focused on the efficient and effective separation of materials (e.g., oils or other chemicals) of interest from a wide variety of liquid sources of oil, oil comprising water, or even organisms and/or cells. More specifically, the present invention relates to the production of commercially valuable products and their isolation using coalescence with a membrane contactor. In non-limiting examples, the liquid source of oil is a biological or non-biological source of oil, e.g., water that is contaminated with oil, oil that is contaminated with water, produced water, mud used in oil and gas extraction, wastewater, tailings pond water, waste oil, any liquid waste or byproducts, from oil and/or gas production, transport, or processing.

In one embodiment, the present invention includes a method of recovering one or more oils from a liquid source using one or more membranes or membrane contactors, comprising the steps of: processing the liquid source by at least one of dewatering, harvesting, or concentrating the liquid source; pumping the processed liquid source, wherein the liquid source comprises the one or more oils to one or more membranes or membrane contactors, wherein the liquid source does not contain an amount of solvent sufficient to disperse the oils; contacting the liquid source with a first surface of the one or more membranes or membrane contactors; coalescing the one or more oils onto the first surface of the one or more membrane contactors; and removing a stream of oil coalesced from the second surface of the one or more membranes or membrane contactors. In one aspect, the method further comprises the step of lysing the source of oil if the source of oil is a biological source. In another aspect, if the liquid source is biological, the step of dewatering, harvesting, or concentrating the biological material is selected from at least one of a pH sweep, resin concentration, electrowicking, centrifugation, filtration, chemical flocculation, chemical sedimentation, bioflocculation, dissolved air flotation, gravity separation, filter press or auto flocculation. In another aspect, the step of lysing the biological source is defined further as selected from at least one of high pressure homogenization, ultrasonic lysing, bead milling or beating, microwave, osmotic shock, chemical lysing, enzymatic lysing, French press, freeze-thawing, apoptosis or drying. In another aspect, the method further comprises the step of processing the coalesed oil to isolate one or more products. In another aspect, the step of processing the coalesced oil is defined further as selected from at least one of winterization, solvent extraction, chromatography, filtration, expeller, French press, supercritical fluid extraction, thermochemical conversion, which may include hydrothermal liquifaction, gasification, pyrolysis, enzymatic conversion, liquefaction, distillation, or catalytic conversion. In another aspect, the step of processing the coalesced oil is defined further as isolating one or more secondary products selected from at least one of astaxanthin, b-carotene, essential fatty acids, polyunsaturated fatty acids, phycobiliproteins, lutein, glycerol, monoglycerides, diglycerides, triglycerides, eicosapentaenoic acid (EPA), docosohexaenoic acid (DHA), arachidonic acid, co-enzyme Q10, phospholipids, phytolipids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, steroids, sterols, or sterol-containing metabolites. In another aspect, the source of oil is a bacteria, an algae, or a cyanobacteria. If the source of oil is biological, in one non-limiting embodiment, the biological source of oils is selected from at least one of Nannochloropsis, Chlorella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora, or Ochromonas. In a related aspect the microalgal species are selected from the group consisting of Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphoracoffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp.,Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorellakessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var. Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Desmodesmus, Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera Effipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis off galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsissalina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheriaacidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana. In another aspect, the biomass is separated and processed into fertilizer, food, energy, animal feed, fish food, human food, pharmaceuticals, or nutraceuticals. In another aspect, the liquid source comprises non-biological sources of oil, selected from at least one of water that is contaminated with oil, oil that is contaminated with water, produced water, mud used in oil and gas extraction, wastewater, tailings pond water, waste liquid waste or byproducts.

In another embodiment, the present invention includes a method of recovering one or more soluble or insoluble oils from a biological source using one or more membranes or membrane contactors, comprising the steps of: lysing the biological source source of oil to form a liquid source of oil and a biomass; pumping the liquid source of oils to one or more membranes or membrane contactors, wherein the liquid source does not contain an amount of solvent sufficient to disperse the oils; contacting the liquid source with a first surface of the one or more membranes or membrane contactors; coalescing the one or more oils within the liquid source onto the first surface of the one or more membrane contactors; and removing a first stream of oil coalesced from the second surface of the one or more membranes or membrane contactors. In one aspect, the method further comprises the step of pre-processing a source of oil by at least one of dewatermg, harvesting, or concentrating the source of oil into a liquid source of oil, wherein the dewatering, harvesting, or concentrating is defined further as selected from at least one of a pH sweep, resin concentration, electrowicking, centrifugation, filtration, chemical flocculation, chemical sedimentation, bioflocculation, dissolved air flotation, gravity separation, filter press or autoflocculation. In another aspect, the step of lysing the biological source is defined further as selected from at least one of high pressure homogenization, ultrasonic lysing, bead milling or beating, microwave, osmotic shock, chemical lysing, enzymatic lysing, French press, freeze-thawing, apoptosis or drying. In another aspect, the method further comprises the step of wherein the step of post-processing the coalesed oils into one or more secondary products is defined further as selected from at least one of winterization, solvent extraction, chromatography, filtration, expeller, French press, supercritical fluid extraction, hydrothermal liquifaction, thermochemical conversion, gasification, pyrolysis, enzymatic conversion, liquefaction, distillation, or catalytic conversion. In another aspect, the liquid source comprises non-biological sources of oil, selected from at least one of: water that is contaminated with oil, oil that is contaminated with water, produced water, mud used in oil and gas extraction, wastewater, tailings pond water, waste oil, liquid waste or byproducts. In another aspect, if the source of oil is a biological material, then the source can be, e.g., a bacteria, an algae, a cyanobacteria, a yeast, a plant, or an animal. In another aspect, the biomass is separated and processed into fertilizer, food, energy, animal feed, fish food, human food, pharmaceuticals, or nutraceuticals. Yet another embodiment of the present invention includes a method of recovering one or more soluble or insoluble oils from a liquid source using one or more membranes or membrane contactors, comprising the steps of: pumping the liquid source comprising the one or more oils to one or more membranes or membrane contactors, wherein the liquid source does not contain an amount of solvent sufficient to disperse the oils; contacting the liquid source with a first surface of the one or more membranes or membrane contactors; coalescing the one or more oils within the liquid source onto the first surface of the one or more membrane contactors; removing a first stream of oil coalesced from the second surface of the one or more membranes or membrane contactors; and processing the coalesced oil is into one or more secondary products. In one aspect, the step of processing the coalesed oils into one or more secondary products is defined further as selected from at least one of winterization, solvent extraction, chromatography, filtration, expeller, French press, supercritical fluid extraction, hydrothermal liquifaction, thermochemical conversion, gasification, pyrolysis, enzymatic conversion, liquefaction, distillation, or catalytic conversion. In another aspect, the method further comprises the step of lysing the source of oil if the source of oil is a biological source into the liquid source. In another aspect, the method In another aspect, the further comprises the step of dewatering, harvesting, or concentrating the liquid source from a biomass, wherein the step of dewatering, harvesting, or concentrating is defined further as selected from at least one of a pH sweep, resin concentration, electrowicking, centrifugation, filtration, chemical flocculation, chemical sedimentation, bioflocculation, dissolved air flotation, filter press, gravity separation or autoflocculation. In another aspect, the step of lysing the biological source is defined further as selected from at least one of high pressure homogenization, ultrasolic lysing, bead milling or beating, microwave, osmotic shock, chemical lysing, enzymatic lysing, freeze press, freeze- thawing, apoptosis or drying. In another aspect, the one or more secondary products are selected from at least one of astaxanthin, b-carotene, essential fatty acids, polyunsaturated fatty acids, phycobiliproteins, lutein, glycerol, monoglycerides, diglycerides, triglycerides, eicosapentaenoic acid (EPA), docosohexaenoic acid (DHA), arachidonic acid, co-enzyme Q10, phospholipids, phytolipids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, steroids, sterols, or sterol-containing metabolites. In another aspect, the source of oil is a bacteria, an algae, a cyanobacteria, a yeast, a plant or animal. In another aspect, the biomass is separated and processed into fertilizer, food, energy, animal feed, fish food, human food, pharmaceuticals, or nutraceuticals.

In another embodiment, the present invention includes a method of recovering one or more soluble or insoluble oils from a liquid source using one or more membranes or membrane contactors, comprising the steps of: obtaining a source of oil from a biomass by at least one of harvesting or lysing the source of oil into a liquid source of oil; pumping the liquid source comprising the one or more oils to one or more membranes or membrane contactors, wherein the liquid source does not contain an amount of solvent sufficient to disperse the oils; contacting the liquid source with a first surface of the one or more membranes or membrane contactors; coalescing the one or more oils within the liquid source onto the first surface of the one or more membrane contactors; removing a first stream of oil coalesced from the second surface of the one or more membranes or membrane contactors; and processing the coalesced oil is into one or more secondary products. In one aspect, the method further comprises the step of repeating the steps of obtaining the oil or coalesing the oil after the first processing step to further purify the oil or obtain one or more secondary products. In another aspect, the one or more secondary products are selected from at least one of astaxanthin, b-carotene, essential fatty acids, polyunsaturated fatty acids, phycobiliproteins, lutein, glycerol, monoglycerides, diglycerides, triglycerides, eicosapentaenoic acid (EPA), docosohexaenoic acid (DHA), arachidonic acid, co-enzyme Q10, phospholipids, phytolipids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, steroids, sterols, or sterol-containing metabolites. In another aspect, the step of harvesting the oil is defined further as comprising at least one of dewatering, harvesting, or concentrating is defined further as selected from at least one of a pH sweep, resin concentration, electrowicking, centrifugation, filtration, chemical flocculation, chemical sedimentation, bioflocculation, dissolved air flotation, filter press, gravity separation or autoflocculation. In another aspect, the step of lysing the biological source is defined further as selected from at least one of high pressure homogenization, ultrasonic lysing, bead milling or beating, microwave, osmotic shock, chemical lysing, enzymatic lysing, freeze press, freeze-thawing, apoptosis or drying. In another aspect, the step of processing the coalesced oil is defined further as selected from at least one of winterization, solvent extraction, chromatography, filtration, expeller, French press, supercritical fluid extraction, hydrothermal liquifaction, thermochemical conversion, gasification, pyrolysis, enzymatic conversion, liquefaction, distillation, or catalytic conversion. In another aspect, the source of oil is a bacteria, an algae, or a cyanobacteria In another aspect, the biomass is separated and processed into fertilizer, food, energy, animal feed, fish food, human food, pharmaceuticals, or nutraceuticals.

In one embodiment, the present invention includes a modular mobile unit on a transportable platform for processing one or more biological cells to yield an oil comprising: one or more de watering/harvesting units to concentrate the one or more biological cells suspended in a medium by removal of the medium, wherein the medium comprises fresh water, salt water, brackish water, growth medium, culture medium or combinations thereof; one or more lysing units to lyse the one or more biological cells, wherein the lysis results in a release of one or more cellular components comprising oils, neutral lipids, proteins, triglycerides, sugars or combinations and modifications thereof from the biological cells; one or more separations unit to separate the released oils and lipids from the medium resulting in a generation of a residual biomass; one or more power supply units to provide electricity to run the dewatering, lysing, and separations units and to remotely operate the unit; one or more control panels to operate and monitor the performance of the dewatering, lysing, and separations units; and at least one of pumping equipment, heat exchangers, distilling equipment, reboilers, condensors for processing the algal cells or the oil. In one aspect, the unit may further comprise at least one of: one or more conversion units to convert the oils; one or more processing units to process the residual biomass for disposal or for conversion to other products; and one or more storage tanks, vessels or containers to store released and separated cellular components or the processed biodiesel or biofuel.

In another embodiment, the present invention includes a method of recovering one or more insoluble oils from a liquid source water that is contaminated with oil, oil that is contaminated with water, produced water, mud used in oil and gas extraction, wastewater, tailings pond water, waste oil, liquid waste or byproducts using one or more membranes or membrane contactors, comprising the steps of: pumping the liquid source comprising the one or more oils to one or more membranes or membrane contactors, wherein the liquid source does not contain an amount of solvent sufficient to disperse the oils; contacting the liquid source with a first surface of the one or more membranes or membrane contactors; coalescing the one or more oils within the liquid source onto the first surface of the one or more membranes or one or more membrane contactors; removing a first stream of oil coalesced from the second surface of the one or more membranes or membrane contactors; and processing the coalesced oil into one or more secondary products.

In another embodiment, the present invention includes a modular mobile unit on a transportable platform for processing an oil comprising: one or more dewatering units and a medium that comprises non-biological sources of oil, water that is contaminated with oil, oil that is contaminated with water, produced water, mud used in oil and gas extraction, wastewater, tailings pond water, waste oil, any liquid waste or byproducts, from oil and/or gas production, transport, or processing; one or more separation units to separate the released oils and lipids from the medium, wherein the separation units comprise one or more membranes or membrane contactors, wherein the liquid source does not contain an amount of solvent sufficient to disperse the oils; contacting the liquid source with a first surface of the one or more membranes or membrane contactors; coalescing the one or more oils onto the first surface of the one or more membranes or one or more membrane contactors; and; one or more power supply units to provide electricity to run the dewatering and separations units and to remotely operate the unit; and one or more control panels to operate and monitor the performance of the dewatering and separations units.

In another embodiment, the present invention includes a method and modular mobile unit on a transportable platform for processing an oil comprising: one or more separation units to separate non- biological sources of oil, water that is contaminated with oil, oil that is contaminated with water, produced water, mud used in oil and gas extraction, wastewater, tailings pond water, waste oil, any liquid waste or byproducts, wherein the separation units comprise one or more membranes or membrane contactors; contacting the liquid source with a first surface of the one or more membranes or membrane contactors; coalescing the one or more oils onto the first surface of the one or more membranes or one or more membrane contactors; one or more power supply units to provide electricity to run the one or more separations units and to operate the unit; and one or more control panels to operate and monitor the performance of the one or more separations units. In one aspect, the liquid source does not contain an amount of solvent sufficient to disperse the oils. BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

FIG. 1 is a process flow that includes the membrane separation technology of the present invention, while the figures shows cultivation as one of the sources of oil, for non-biological sources of oil the user would skip to the last step of processing.

FIG. 2 is a flow diagram of a membrane contactor system for use with the present invention for any source of oil.

FIG. 3 is a diagram of the layout of the trailer described in the present invention; and

FIGS. 4A and Figure 4B show a top view and a side view of the trailer shown in FIG. 3. Fig. 4C is a schematic illustration showing the different elements of the mobile photo bioreactor of the present invention. FIG. 4D is similar to FIG. 4C with the trailer closed. FIG. 4E is an alternative embodiment of the mobile photobioreactor of the present invention specifically designed if the baffles cannot mitigate possible adverse effects of acceleration and deceleration on productivity.

FIGS. 5 to 10 show various flowcharts that summarize the various methods of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

There has been significant effort in recent decades to produce "food, feed, fiber, fresh water, and fuel using microorganisms" (a phrase coined by Michael E. Webber, PhD, University of Texas at However, the production of food, feed, fiber, fresh water, or fuel from microorganisms (such as algae) requires multiple steps. Various interchangeable technologies can be used to accomplish each step. The present inventors show herein the ability of hollow fiber membrane separation technology (initially developed for gas separation), in conjunction with separation technologies both upstream and downstream, in e.g., the inventors' U.S. Patent Nos. 8,486,267, 8,491,792, 8,617,396, and 9,149,772, relevant portions incorporated herein by reference. The present invention is directed to the integration of separation and post-processing technology in any production process to produce valuable products. The present inventors show herein the ability of hollow fiber membrane separation technology, developed for gas separation. This gas separation technology is used in conjunction with many upstream and/or downstream techniques and technologies. The present invention is directed to the integration of separation and post-processing technology in any production process to produce valuable products.

The present invention describes a variety of processes that are part of the recovery of various useful materials obtained from a variety of sources that include upstream processes, the processing of the liquids and other materials to recover water or oil, and processes that are downstream from the recovery step. Using a membrane contactor the present invention can be used as a standalone complete process, or can be connected to other technologies and existing equipment both upstream and downstream from the coalescence of material. The upstream processes include but are not limited to growing, harvesting, concentrating, and/or lysing, certain biological and other materials to release the useful materials. The next stage involves separating the various components of the source of materials from the actual material using a membrane contactor. Finally, certain processes downstream from the membrane contactor are described and claimed herein before or after coalescing the material (whether as exclusion or passage through the membrane contactor).

As used herein, the term "membrane", "membrane contactor", or non-dispersive contactor refers to a hydrophobic microporous hollow fiber membrane. Non-limiting examples of membrane contactors include hydrophobic membrane or membrane contactors or membrane contactor modules that comprises hollow fiber microporous membranes, e.g., hydrophobic hollow fiber membrane made from polyethylene, polypropylene, polyolefins, polyvinyl chloride (PVC), amorphous Polyethylene terephthalate (PET), polyolefin copolymers, poly(etheretherketone) type polymers, surface modified polymers, mixtures or combinations thereof.

The skilled artisan will recognize that the term "liquid source" or "feed source" refers to a liquid, a colloid, a liquid-solid or other liquid fluids that that have oil for separation using the present invention. Non-limiting examples of a liquid sources of oil include, but are not limited to, a water-based biological medium comprising an oil (with or without the biological organism), and non-biological sources of oil such as: oil-in-water, water-in-oil, oil-rich streams, crude oil containing liquids, or aqueous streams, produced water, transportation fuel, heating oil, refined petroleum products, petrochemicals, reclaimed oils, waste oils, oil industry liquid streams, oil contaminated water or brine, drilling mud, and oil sands tailings, tailings pond water, waste oil, any liquid waste or byproducts, from oil and/or gas production, transport, or processing. For example, "contaminated oil" can be an oil that is an "off-spec" oil or fuels that can not be sold for the same price, and many require clean up prior to sale or can be found in, e.g., produced water that is contaminated with oil, e.g., crude oil. Because "off-spec" oil has diminished value, the present invention has immediate use in oil clean up, with diverse, large commercial applications. Produced water refers to byproducts of the oil industry that is water, connate water, brine, or the like, that includes byproducts of oil and/or gas extraction. The present invention can be used with water produced at the wellhead, or from evaporation ponds. In some cases the produced water is found naturally in the formation, and the oil and/or gas is extracted along with the water. In other cases the produced water may be the byproduct of injection wells, in which water is injected into the formation or reservoir to increase oil and/or gas production. More generally, the term "oil" refers to, e.g., hydrocarbon or hydrocarbon-rich molecules that are found or recovered individually, or as a complex mixture, that includes but are not limited to crude oil, fractions or crude oil, petrochemicals, lipids, hydrocarbons, fatty acids, triglycerides, aldehydes, etc.

Non-limiting examples of biological sources of materials that would be considered for separation in the present invention would be phototropic, heterotrophic, and mixotrophic biological growth chambers, such as open ponds, photobioreactors, and fermenters, as well as other methods of creating aqueous streams with oil (e.g., hydrocarbons, oils or other chemicals whether from a biological source or not) contained in the aqueous stream (e.g., an olive press which may produce a mixture of olive oil and water). Non-limiting examples of biological sources, specifically cells, can include but are not limited to algal, fungal (yeast and other fungi), bacterial, helminthic, cyanobacterial, plant, animal oils and fats, insect cells or driven by any of a variety of viruses or extranuclear nucleic acids that trigger expression in a living organism. These aqueous streams can be upstream processed by non-limiting examples such as dewatering, harvesting, and/or other ways of concentrating the biological material from at least one of a pH sweep, resin concentration, electrowicking, centrifugation, filtration, chemical flocculation, chemical sedimentation, bioflocculation, dissolved air flotation, filter press, and/or autoflocculation. In some cases, none of these concentration technologies are necessary. Non-limiting examples of further upstream processing to obtain the materials include lysing a biological source by at least one of high pressure homogenization, ultrasonic lysing, bead milling or beating, microwave, osmotic shock, chemical lysing, enzymatic lysing, French press, freeze-thawing, or drying. In some cases, none of these technologies are necessary. These upstream processing steps, and others that have not been specified, serve to prepare the aqueous stream for processing in the present invention.

Non-limiting examples of processing steps that can be used prior to, concurrently with, or after coalescing the oil with the membrane contactor can be at least one of winterization, solvent extraction, chromatography, filtration, expeller, French press, supercritical fluid extraction, hydro thermal liquifaction, thermochemical conversion, gasification, pyrolysis, enzymatic conversion, liquefaction, distillation, or catalytic conversion. The combination of upstream and downstream processes with the coalescence of material(s) using the membrane contactor provides several distinct advantages, including but not limited to: (i) continuous, automated flow-through process; (ii) low operating cost; and (iii) specific materials may be preferentially selected by use of specific solvents or oils. Another advantage is that it is possible using the present invention to recover living biomass for continuous growth, production and use. As used here, the term "mobile" refers to any vehicular configuration that permits the transportation of a unit as described herein, be it on land, sea or air. In certain embodiments the unit can be placed on a pallet, in or on a container or skid, in or on a trailer, and may include, e.g., permanently or non- permanently attached: wheels, runners, skids, skis, ball-bearings, tracks, air bearings, skirts (e.g., for hovering on air or water, or rollers. The skilled artisan will recognize that the mounting and transportation of the unit will depend on the weight of the unit, the availability of one or more of the following non-limiting examples: water, electrical, electromagnetic, hydraulic, pneumatic, sun, wind, mechanical or other source power, for powering the operations of the unit.

As used herein, the term "winterization" refers to the enrichment of fatty acids. Winterization relies upon the difference in melting points between saturated and unsaturated fatty acids to separate oil into enriched fractions. For example, crystals are grown or formed when the temperature of the molten fats and oil (or its solution) is lowered, e.g., by rapid chilling or slow cooling (depending on the wax or fat). The solubility at the final or separation temperature determines the composition of the crystals formed as well as their mother liquor. The next step is separation of the solidified fats or oils, e.g., using filtration, vacuum filtration, centrifugal separation, conical centrifugation, hydraulic presses, membrane filter presses, and/or decanters. For example, a simple winterization step can be used in fish oil processing to generally remove saturated fatty acids and leave behind the bulk of the unsaturated fats, e.g., high value, Omega3 rich, fatty acids. In another non-limiting example, winterization can be used to separate fuel grade oils from nutraceutical grade oils. At scale, it is often more practical to separate components by winterization, e.g., when it is impractical to separate by distillation.

Furthermore, in various embodiments the invention utilizes a mobile, transportable, or stationary apparatus to process materials. The processing unit can be self-contained and fully integrated to process oils. If the source of oil is biological, it can begin with harvesting, concentration, lysing, oil separation, and downstream processing processes, thereby greatly reducing operational costs. Broadly, the present invention can includes two basic steps: (1) concentrating the starting material, e.g., removing clarified water from the slurry by settling, centrifugation, filtration, etc., if applicable; (2) recovering relevant materials (oils, chemicals, or lipids) producing a lipid stream and a non-lipid stream that can be further processed. In one non-limiting example, the process steps can include at least one of: (1) harvesting any source of materials; e.g., the source could be growing reservoirs (e.g., bioreactors, ponds, tanks, fermenters), oil ponds, produced water or other like reservoirs that include materials to be coalesced with the membrane contactor; (2) concentrating the materials, e.g., removing clarified water from the slurry by settling, centrifugation, filtration, etc., if applicable; (3) if the source of material is a living cell lysing the living cells (or completing the lysing of partially lysed or dead cells); and/or (4) recovering relevant materials (oils, chemicals, including lipids, from the lysed cells or from the un-lysed stream) producing a lipid stream and a non-lipid stream that can be further processed.

Various technologies for use with the present invention are described in detail herein below and may be used individually or in combination with the present invention. For example, any of a wide variety of upstream technologies or methods can be used to obtain a liquid source of oil, which are then input into the membrane contact coalescence system taught herein. Also, a wide variety of downstream processing technologies or methods can be used with the membrane contact coalescence system taught herein. Thus, the skilled artisan will recognize, based on the non-limiting examples provided herein below, that the coalescence with a membrane contactor can be used in conjunction with various upstream and/or downstream processing devices, methods and/or systems to separate useful materials from a liquid using established technologies but at a greatly reduced operational cost and with greater yields and efficiency as disclosed herein. Furthermore, the present invention can be used to process various liquid streams from a variety of sources. In fact, it is one of the distinct advantages of the present invention that it can be used with a wide variety of upstream and downstream processes and streams.

For those examples that includes the isolation of materials from a biological source, media and/or growth conditions can be used with any living cell (including those that are transformed, transfected, transgenic and/or virally infected) to optimize production of a material for isolation and/or postprocessing using the present invention. The skilled artisan using conventional methods and reagents knows how to grow living cells, and which methods can be modified to meet or maximize the requirements of that particular cell. In one specific non-limiting example using plants, algae or cyanobacteria species (for example those in the group diatoms (bacillariophytes), green algae (chlorophytes), golden-brown algae (chrysophytes), red algae (rhodophytes), and blue-green algae (cyanophytes)), these cells can be cultured or grown phototrophically (under sunlight in flasks, bioreactors, open or closed ponds), heterotrophically (in the dark in flasks, bioreactors, or fermenters using a carbon source as a feedstock) or mixotrophically (using a mix of different energy and carbon sources), or any combination of growing techniques for the purpose of enhancing algal or cyanobacterial lipid, carbohydrate or protein content. In the specific case of algae or cyanobacteria, the unit may process densities in the range of 100 mg/L - 150 g/L.

If necessary, the cells can be initially harvested or isolated using a wide variety of technologies, such as dewatering, harvesting, or concentrating by, for example a pH sweep, resin concentration, electrowicking, centrifugation, filtration, chemical flocculation, chemical sedimentation, bioflocculation, dissolved air flotation, filter press or autoflocculation, or gravity separation. In one specific, non-limiting example, plate settlers are used in water and wastewater clarification applications to increase the rate and efficiency of clarification. For the purpose of dewatering the algal or similar cells, a plurality of tube and/or plate settlers are positioned in a slanted or inclined position, preferably fifty-five to sixty-one degrees, in fluid in the clarifier. Cell culture water is passed upward in the clarifier through the tube and/or plate settlers so that cells are collected on the settlers, and via gravity slide down to the lower portion of the settler, while clarified water is completely passed through the settlers. This process results in removal of 1 - 99.9% of the water, producing a cellular slurry.

The present invention can use a process to produce a deflocculated algae or biomass concentrate from dilute aqueous solutions. The biomass resulting from the process of the present invention may be processed into a liquid biofuel or into other products that can utilize the biomass including animal feed, biogas (methane generation) or platform chemical production.

Again, for the example of a biological source of oil, the invention can use two processes in series, e.g., flocculation of the algae to remove it from the feed water followed by deflocculation to separate the algae from the precipitated solids. A continuous-feed flocculation process can be used to achieve separation by adding lime or other base (e.g., NaOH) to the feed solution to rapidly raise the pH of the aqueous solution. The addition of ions such as Mg or Ca may be required depending on the composition of the background water. For example, if the quality of the water stream is not conducive for optimal flocculation pretreatment may be required, e.g., if the water is hard and has a high alkalinity, the water may be pre -treated by addition of acid and air sparging, prior to the precipitation process. The rapid pH rise in the main process leads to precipitation of the inorganic constituents in the feed water and association of the microalgae with the precipitate. Release of the biomass requires dissolution of the precipitate, which is facilitated through pH reduction via carbon dioxide or other acid such as HC1. In another embodiment of the invention, base addition modifies the surface charge characteristics of microalgae and causes the biomass to flocculate with minimal formation of inorganic precipitate. In this scenario, low Mg and Ca concentrations are required in the water. In either case, the flocculated cells (e.g., bacterial, cyanobacteria, algae, yeast, plant, etc.) enmeshed in the inorganic precipitate settles rapidly to the bottom of a continuous flow plate, filter plate, or tube settler. The cells are thus removed from the feed solution. In certain operating modes, a stream of flocculated algae can be recirculated into the feed tank to promote faster and more efficient flocculation of dilute cells. The treated effluent water is suitable (after pH adjustment) for discharge and potentially for recycle to the growth pond. The cells enmeshed in the inorganic precipitate (or flocculated) are deflocculated in a continuous flow deflocculation process that utilizes contact with carbon dioxide or other acid to reacidify the precipitated solids and release the microalgae or other biomass. The resulting product is a homogenous slurry of biomass that has been recovered from the feed solution. Release of the algae from the precipitated solids can be enhanced by mechanical agitation.

The total harvested cellular slurry volume can be fed by pump or gravity flow to a cellular lysis device, method or system. Non-limiting examples of cellular lysis include but are not limited to, high pressure homogenization, ultrasonic lysing, bead milling or beating, microwave, osmotic shock, chemical lysing, enzymatic lysing, freeze press, freeze-thawing, apoptosis or drying. In one specific non-limiting example, electromechanical lysis can be conducted using a chamber as a preparatory process for use in cellular oil separation. The chamber provides a method for the electrical treatment of one or more biological cells suspended or surrounded by a lysing medium comprising of fresh water, a salt water, a brackish water, a growth medium, a culture medium or combinations thereof wherein the electrical conductivity of the lysing medium is different from the electrical conductivity of a cell membrane and the cytoplasm of one or more biological cells. The cellular lysis exposes cellular components that comprise the materials isolated herein, and can include, e.g., oils, lipids, fatty acids, proteins, carbohydrates, or combinations thereof. The lysis and other processes for use in conjunction with the present invention have been previously described in detail in by some or all of the present inventors in U.S. Patent No. 8,673,623, Patent Applications Serial Nos. 12/233,742, 13/186,282, and/or 13/439,340; or U.S. Patent Application Publication No. US 2011/0107655 by Kempkes et al., relevant portions of which are incorporated herein by reference.

The present inventors in US Patent Nos. 8,486,267 B2; 8,491,792 B2; and 8,617,396 B2, and related cases, relevant portions incorporated herein by reference) demonstrate the separation of oil from a liquid source through the use of a hydrophobic hollow fiber membrane. In a brief example of oil produced from a biological source, the lysed cell and/or cell debris concentrate is fed on the shell side while solvent or an oil surrogate (e.g., oil that is algal, plant, fungal, etc.) is fed on the fiber side, although a collection fluid is not needed. The solvent or surrogate oil acts as a sweep and recovers the coalesced oil within the tube surface of the hollow fibers. A natural fatty acid may be added to the lysed cell concentrate to minimize fouling on the outer surface of the fiber and increase oil coalescence. The one or more counter- flowing solvents comprise non-polar solvents, alkanes such as hexane and heptanes, aromatic solvents such as benzene, toluene, ethers such as diethyl ether, halogenated solvents such as chloroform, dichloromethane, and esters such as ethyl acetate. The counter-flowing solution may also comprise of surrogate oils such as plant, seed or nut oils, components of biodiesels selected from monoglycerides, diglycerides, triglycerides, fatty acids, and fatty acid methyl esters.

The process described herein results in a lipid stream and a remaining material (e.g., a biomass stream) that may be further processed for relevant bioactive chemicals or used for other applications. Non-limiting examples of downstream processes include but are not limited to solvent extraction, chromatography, filtration, expeller, French press, filter press, supercritical fluid extraction, hydrothermal liquifaction, thermochemical conversion, gasification, pyrolysis, enzymatic conversion, liquefaction, distillation, or catalytic conversion. Biological cells for use with the present invention include those from a division such as Cyanophyta, Archaeplastida/Plantae sensu lato (includes the Phylum Viridiplantae, plants, which includes Chlorophyta, Rhodophyta, and Glaucophyta); Cabozoa (includes the Kingdom Excavata and Supergroup Rhizaria that represents the Euglenophyta and Chlorarachniophyta); Chromaveolata (includes the Supergroup Chromista and Superphylum Aveolata that represent the Heterokontophyta, Haptophyta, Cryptophyta, and Dinophyta), as well as the Kingdom Fungi (all yeasts and fungal-related organisms). Moreover, the one or more biological cells can be, but are not limited to, plant extracts, seed extracts, fruit extracts, vegetable extracts, leaf extracts, seedling extracts, algal cells, bacterial cells, fungal cells (including yeast), insect cells, helminthic cells, plant cells, animal cells, mammalian cells, and/or cells that have been transformed, transfected, transgenic, knock-in, knock-out, conditionally transgenic, knocked- in, knocked-out, virally infected cells or combinations thereof. In one non-limiting example, the process disclosed herein can be used to recover citrus oil that is squeezed out of, for example, limes or lemons, grapes, grapefruit, or oranges, olive oil, nut oil, and other natural oils. As one non-limiting example of cells that can be used with the present invention, a wide variety of algal cells are described specifically. Equivalent lists of cells that are of bacterial, cyanobacterial, plant, insect, helminthic, animal, mammalian, or other cells, are incorporated herein by reference as will be known to the skilled artisan. As used herein the term "algae" represents a large, heterogeneous group of primitive photosynthetic organisms, which occur throughout all types of aquatic habitats and moist terrestrial environments. For use with the present invention, examples of algae include but are not limited to any of the diatoms (bacillariophytes), green algae (chlorophytes), blue-green algae (cyanophytes), golden-brown algae (chrysophytes), red algae (Rhodophyta), haptophytes, freshwater algae, saltwater algae. In another aspect one or more algal cells comprise microalgae selected from a class comprising Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae. In yet another aspect the microalgal genera are selected from the group consisting of Nannochloropsis, Chlorella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora, and Ochromonas. In a related aspect the microalgal species are selected from the group consisting of Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphoracoffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp.,Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorellakessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var. Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Desmodesmus, Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera Effipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis off. galbana, Ilsochrysis galbana, Lepocinclis, Micr actinium, Micr actinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsissalina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheriaacidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis,Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

The present invention provides a number of novel features that differentiate with currently employed methods and technologies, e.g., (i) lysis and separation of lipids including Omega fatty acids on a continuous or batch basis in a flow-through system; (ii) the lysis is performed without the use of chemicals; and (iii) the Omega fatty acids are extracted via membrane technology whereby the fatty acids are coalesced into a solvent or solvent-less solution while the biomass does not contact solvent.

The present invention also addresses some of the problems associated with methods currently in use for recovering oils from cellular cultures, for example: (i) automated processing of cell derived lipids (e.g., omega or other fatty acids or lipids); (ii) oils are recovered in a solvent or solvent-less solution; and/or (iii) residual biomass is uncorrupted by solvent or other chemicals.

In addition to the extraction of oils the process of the present invention can be adapted for the separation of commercially valuable proteins and carbohydrates and is amenable for commercialization without significant modifications or changes.

Using the present invention, the inventors have been able to produce a wide variety of products starting from a wide variety of sources to produce, for example: (1) bio-oil(s) as an intermediary for fungible fuel production (e.g., biodiesel, renewable diesel, jet fuel, synthetic gasoline, and other hydrocarbon fuels); (2) biomass for conversion to fuel (e.g., via anaerobic digestion, combustion, or thermochemical conversion); (3) food and nutritional products produced from the bio-oil or the biomass (e.g., protein for human consumption, omega-3 fatty acids, fiber, and astaxanthin); (4) animal feed (e.g., protein meal to replace corn meal, soy mean, or fish meal); or (5) other industrial products produced from the bio-oil or the biomass (e.g., fertilizer, pigments, chemicals and cosmetics).

The production pathways required to produce these products are varied and many of the steps are interchangeable. The basic steps in the process are common to all processes and all end-products: cultivation of the microorganism, processing of the biomass, and refinement of the biological constituents into marketable products including food, feed, fiber, freshwater and fuel products.

The present inventors use herein a number of separations technologies that are designed to partition a wide variety of microorganism constituents. The inventors have successfully tested this process with several interchangeable upstream processing options, and processes for production of the downstream products, known in the industry, but heretofore not used in combination with the surprising and unexpected results demonstrated herein. Additionally, the inventors have performed testing on certain downstream products as described below. The inventors demonstrate herein the efficacy of integrating the separations technology with interchangeable upstream technologies as well as the ability to use the separations technology to selectively extract a range of down-stream products or precursors to downstream products. As such, the present invention provides for the first time a process for using a separation technology in conjunction with any upstream production methods (including but not limited to: cultivation of microorganisms in ponds or cultivation in bioreactors; collection of biomass by any harvesting and/or dewatering method such as centrifugation, filtering, or flocculation; and cell disruption technologies such as electromechanical lysing, solvent permeability, ultrasound, or mechanical cell disruption) for a range of marketable downstream biological constituents (including, but not limited to: (1) bio-oil as an intermediary for fungible fuel production (e.g., biodiesel, renewable diesel, jet fuel, synthetic gasoline, and other hydrocarbon fuels); (2) biomass for conversion to fuel (e.g., via anaerobic digestion or thermo chemical conversion); (3) food and nutritional products produced from the bio-oil or the biomass (e.g., protein for human consumption, omega-3 fatty acids, fiber, and astaxanthin); (4) animal feed (e.g., protein meal to replace corn meal, soy mean, or fish meal); or (5) other industrial products produced from the bio-oil or the biomass (e.g., fertilizer, pigments, chemicals and cosmetics).

The present invention can be integrated with various, known upstream and downstream processing technologies, similar to the cultivation, processing, and refinement of agricultural products such as corn, soybeans, and livestock products. For example, corn (of various genera) is cultivated using various cultivation technologies such as greenhouse cultivation, drip irrigation, flood irrigation, and non-irrigated cultivation. The corn is then harvested via one of several methods including hand-harvesting and machine-harvesting. After the corn biomass is collected, it is fractionated and refined into various products such as food (e.g., raw corn, corn flour, corn syrup), animal feed (e.g., kernel products, hominy feed, and gluten feed), fuel (e.g., ethanol), and various industrial products (e.g., adhesives, alcohols, and other chemicals). Many of the production technologies for producing corn products are interchangeable. For instance, regardless of whether the corn is cultivated in a greenhouse or in a non-irrigated field, and regardless of whether it is harvested by hand or by a combine, the biological constituents contained in the corn plant can be separated with a range of technologies and the resulting isolated constituents can be refined into a variety of downstream products.

The present inventors demonstrate therein the ability to use the separations technology with multiple species of biological sources (e.g., algae) that were cultivated using multiple technologies (specifically bioreactors, tanks, open and covered ponds, and waste-water treatment ponds, both phototrophic-grown and heterotrophic-grown), harvested using various technologies (including centrifugation, flocculation, filter press, and electro-wicking), processed with various cell disruption methods (including electromechanical lysing, mechanical disruption (e.g., French press), bead-milling, physical disruption (e.g., rapid depressurization), and solvent disruption (e.g., hexane extraction). These upstream processing technologies are interchangeable and the studies demonstrate not only the ability for the separation technology to be used in conjunction with these specific methods, but also can be expanded to other methods.

Furthermore, the inventors demonstrate herein the ability to use the separations technology to recover bio-oil and biomass that can be further processed into a range of intermediate and end-products, such as those mentioned hereinabove, namely: (1) bio-oil; (2) biomass for conversion to fuel; (3) food and nutritional products produced from the bio-oil or the biomass; (4) animal feed or (5) other industrial products produced from the bio-oil or the biomass. The recovery of these downstream products demonstrates not only the ability of the separations technology to produce these specific products, but also other products that originate from biological constituents. The oil released thereby can then be processed by the membrane contactor of the present invention. Finally, the oil coalesced by the membrane contactor can then be post-processed by, e.g., membrane separation, solvent separation, thermochemical conversion or other methods of separation and processing as taught herein below.

Figure 1 is a process flow diagram of methods tested by the present inventors in conjunction with the membrane separation technology and other equivalent interchangeable technologies. Data for each tested upstream and downstream process is discussed in more detail herein below. The top line of Figure 1 shows the various cultivation systems that can be used with the present invention, including but not limited to open pond, covered pond, tanks, bio-reactors, natural growth and other sources of growth. To gather the biological materials or other liquid sources of oil, the present invention can use any of a number of methods to obtain the oil from the liquid source of oil, including but not limited to one or more of the following: centrifugation, flocculation, filtering, dissolved air, natural settling or other harvesting methods or equipment. When the oil is trapped in, e.g., a biological carrier such as bacteria or algae (or equivalents thereof such a micelles, liposomes, etc.), the oils can be released by, e.g., electromechanical lysing, filter press, French press, pressurization, solvent disruption, or other disruption methods such as freeze-thawing.

Figure 2 is a flow diagram of a basic membrane contactor system 200 for use with the present invention. In this embodiment, the membrane contactor 202 is used in one example of a novel oil recovery process of the present invention. The process comprises a membrane contactor 202 comprising a plurality of microporous hollow fiber (MHF) membranes 204 and a central baffle 206. In one non- limiting example of an oil, non-polar cellular oil 208 is fed (pumped) through the hollow membrane fibers 204 and is contacted with the lysed yeast or algal oil concentrate 212 contained in the shell portion of the membrane contactor 202. The non-polar oil 216 coalesces onto the hydrophobic fiber surface of the membrane fibers 204 and dissolves into oil contained in the walls and the counterflowing oil phase 208 and can be removed. There are two exit streams from the contactor 202, a yeast or algal biomass stream 210 which is processed further (dried) a stream 208a which contains the yeast or algal oils and lipids 216 that is collected in a tank 214. Part of the oil 216 can be removed from the tank 214 and fed to the contactor 202 to repeat the process. Media, nutrients, additional organisms, liquid or other compositions can be provided from burettes 219. Multiple pumps and valves may be used to control the flow of the various liquids and components.

Table 1 summarizes the use of the present invention with various upstream processing technology options, all of which were able to provide a significant amount of initial biomass, in this example algal biomass. Table 1 lists the integrated processing experiments, the growth volume from which the algae were harvested, and describes the initial biomass content.

Table 1. Volumes, algal concentration, and biomass yields.

Table 2 lists the harvesting and lysing methods used during the integrated processing experiments. For example, the harvesting efficiency is defined as the amount of harvested dry mass, M _HM , divided by the amount of dry grown algal biomass in the growth medium, M _GM

<P _h arv = 5 ^s * [ ^" ] (1) [0001] The cell lysing efficiency is the lysed dry mass, M _LM , divided by the harvested dry mass,

Table 2. Harvesting efficiency and lysing efficiency for each batch. Electromechanical (EM) lysing was used for all studies (a harvesting efficiency of greater than 1 may indicate a sampling error).

Table 3 presents the amount of biocrude that was recovered during each experiment, all of which used the membrane contactor.

Table 3. Separation methods and the recovered biocrude mass.

In addition to centrifugation, flocculation, and EM lysing, the membrane oil recovery technology has also been used by the inventors in conjunction with alternative interchangeable upstream processing methods such as bead milling, filter press, a pressure-washer, and a French press. It was found that the present invention can be used with a wide variety of both upstream and downstream processing technologies in an efficient and effective manner.

Furthermore, the following technologies can be used upstream or downstream from the present invention. Harvesting, lysing, separation/conversion technologies, and upgrading/refining methods are discussed.

Among others, the present invention can be used in conjunction with the following dewatering, harvesting, or concentrating technologies: pH sweep flocculation, resin concentration, electrowi eking, centrifugation, filtration, chemical flocculation, chemical sedimentation, bioflocculation, dissolved air flotation, filter press, gravity separation and/or auto flocculation.

pH Sweep Flocculation. The pH is increased using Lime, CaOH, or NaOH to -10-12, the algae flocculate, settle out, and the supernatant is removed. The algae can then be de-flocculated using CO ₂ injection.

Resin Concentration. Algae are passed through the resin, they stick to the resin material, and then the resin is flushed with a different material to remove concentrated algae.

Electrowicking. An electric field is placed across the dilute volume, algae are attracted to the positive electrode thereby increasing the concentration, and the concentrated algae can be removed.

Centrifugation. Increased gravity force, achieved by rotation, is used to settle algae. Several types of centrifuges exist.

Filtration. Algae are filtered out of the growth medium, often with assistance from pressure (i.e., a filter press) or a vacuum. Microstrainers are another form of filtration.

Chemical Flocculation and Sedimentation. Chemicals are used that neutralize algal charge to enable flocculation and settling. Flocculants include FeCl ₃ , A1 ₂ (S0 ₄ )3, Fe ₂ (S0 ₄ )3, alum, polyferric sulfate, polyelectrolytes, and commercial polymers (chitosan, Zetag).

Bioflocculation. Cyanobacteria or bacteria (e.g., Paenibacillus sp. AM49) are added to a growth volume and algae settle.

Dissolved Air Flotation (DAF). Tiny bubbles are released into the growth volume, which adhere to the algae and raise them to the surface where they can be recovered. This is a common method used in wastewater treatment.

Gravity Separation. Algae are settled either in the growth volume (e.g., pond) or in a secondary settling reservoir. Autoflocculation. This method has been described as controlling algal motility or interrupting CO ₂ supply causing flocculation. This term has also been used to define pH sweep flocculation and chemical flocculation.

The present invention can also take advantage and can be used with a wide variety of lysing/cell disruption technologies, such as: electromechanical lysing, high pressure homogenization, ultrasonic lysing, bead milling or beating, microwave, osmotic shock, chemical lysing, and drying.

Electromechanical Lysing. A strong, short electric field is applied to cause: 1) electro-distension, 2) electroporation, or 3) initiation of apoptosis.

High Pressure Homogenizer. This method uses pressure to drive an algal slurry through an orifice. The shear forces within the liquid are responsible for "homogenizing" or disrupting the cells. High- pressure homogenization has been used to disrupt Haematococcus, Candida lipolytica, and Pseudomonas aeruginosa.

Ultrasonic Lysing. High frequency sound waves (-10-20 kHz) are applied to inactivate or disrupt cells, which can improve extraction efficiency in some algae. Shock waves resulting from cavitation rupture the cells.

Bead Beater/Mill. Cells are ground by high-speed spinning with small beads. This method has been used to improve extraction yields for oil analysis samples.

Microwave. Algae are exposed to microwaves for improved solvent extraction.

Osmotic Shock. A rapid change in salinity causes an osmotic shock that can rupture cells.

Chemical Lysing. The addition of a chemical, such as acids, alkalis, surfactants, enzymes or solvents lyse cells.

Drying. For some extraction methods it is necessary to dry the algal biomass beyond the post- harvest concentration. Drying can be accomplished by spray drying, freeze drying, drum drying, sun drying, lypholizing, or heating.

There are numerous separation and conversion techniques and the present invention can be used in parallel or in conjunction with these methods, which include: solvent extraction, chromatography, expeller/french press, supercritical fluid extraction, protein extraction, biochemical conversion, enzymatic conversion, and thermochemical conversion.

Solvent Extraction. The algae are exposed to a chemical solvent to extract desired cellular components. The Bligh and Dyer and Folch methods (or variations of them) are commonly used. One specific solvent extraction method is Soxhlet Extraction, named for its inventor, which often uses hexane to extract lipids from dry algal biomass. Isopropyl alcohol (IPA) has been used to extract lipids in wet algae. Solvent extraction often accomplishes cell lysing and component extraction simultaneously. Solvent extraction of algal biomass can also be used to extract astaxanthin, b-carotene, phycobiliproteins, lutein, and fatty acids. Chromatography. Several chromatography methods, including high-pressure liquid chromatography, thin layer chromatography, and gas chromatography, can be applied to separate and quantify different compounds of interest in a feedstock material.

Expeller/French Press. Algae are dried and the oil is pressed out similar to soybean extraction. Supercritical Fluid Extraction. Algae are dried, often crushed, and then exposed to super-critical C0 ₂

(sometimes in addition to methanol), which separates oils from biomass. Generally, supercritical CO ₂ requires dry materials and the use of moderate temperatures and pressures to extract thermolabile compounds.

Protein Extraction. This process may involve diluting the biomass in an aqueous alkaline sol ution and the proteins can then be recovered by centrifugation, and then dried to a powder that is the end- product.

Biochemical or Enzymatic Conversion. Several biochemical conversion processes exist, including anaerobic digestion, fermentation, and biochemical catalytic conversion. In anaerobic digestion - a common process in wastewater treatment - algae are used as feedstock to produce biogas (methane and carbon dioxide). Fermentation converts sugars to desired output products using bacteria or yeast. When catalysts are used in conjunction with microbes to convert feedstock to output products, it is termed catalytic conversion.

Thermochemical Conversion. It is also possible to use the present invention in combination with thermochemical conversion. The most common thermochemical conversion processes are liquefaction, pyrolysis, and gasification. Liquefaction (including hydrothermal liquefaction) converts high molecular weight organic compounds to low molecular weight oils at high temperatures and pressures, and often with the aid of a catalyst. Pyrolysis is the conversion of high molecular weight organic compounds to oil under high temperature without oxygen and under raised pressures. Algae can be gasified using several methods (such as catalytic hydrothermal gasification), and some methods yield syngas that can be used as a feedstock for the Fischer-Tropsch process. The co-products of each thermochemical conversion process vary, and can include gases, aqueous liquids, and solid char.

There are also several downstream processing techniques for upgrading and refining the products produced by the separation/conversion processes, and the present invention can be used in conjunction with these methods, which include: transesterification, enzymatic conversion, catalytic cracking, and hydro-treatment.

Example. Green isolation of nutritional supplements, nutraceuticals and pharmaceuticals.

Numerous products or co-products can be produced from, e.g., bacteria, cyanobacteria, algae, plants, and yeast. These products can be produced from the oil or the biomass or both. Non-limiting examples of these products or co-products include, e.g., Omega 3 and Omega 6 oils. Others include: carotenoids (e.g., astaxanthin), or even organic fertilizers from the biomass. The unique aspect of the isolation of these products or co-products is their isolation using the devices and methods taught herein, because a solvent is not necessary to extract these non-polar or oily products. Nutraceuticals and pharmaceuticals that are sold or advertised, as being "green" must not be produced (\vww.carloerbareagenti.com/Repository/Do\vnload/pdf/Catalog ue/EN/catcheml 00_sez2_green_en.pdf), extracted or isolated with solvent extraction (a non-green process). The present invention also allows the immediate packaging of these products isolated as green without additional processing steps as isolated or combinations of products into pills or capsules.

FIG. 3 shows an isometric view of the mobile algal oil extraction unit 300 for use with the present invention. The unit 300 comprises a trailer bed or a surface 302. The mobile unit is rendered portable by one or more sets of wheels 304. The mobile unit is a part of or can be attached to a trailer for the moving the processing equipment to the algae growth pond or facility. The extraction unit comprises one or more dewatering units 306 to harvest and concentrate the algae by the removal of the water. The unit 300 is also equipped with one or more lysing units 308 the comprise power supplies and cells to provide electromechanical lysing and breakdown the algal cell walls. The unit 300 further comprises one or more separations units 310 that separate water from oil from residual biomass. The unit may also be optionally equipped with one or more power supplies 312 for remote operation. The control panel for the unit 300, is depicted in 314.

FIGS. 4A and 4B provide a top view (4A), and side view (4B) of a mobile algal oil processing unit 300 of the present invention, respectively. FIG. 4C is a schematic illustration showing the different elements of the mobile photo bioreactor 100 placed on a trailer bed or platform 114. The power supply unit 102 comprises an independent diesel generator or equivalent. The bioreactor 100 comprises a nutrient supply 104 as well as heating and cooling units 106. The transparent tubes of growing algae are seen in 112. Light sources for the growth of the algae are shown by 108 and diffusers if needed are shown by 110. FIG. 4D is similar to FIG. 4C with the trailer closed. The mobile photo bioreactor 230 placed on a trailer bed or platform 234. The transparent tubes of growing algae are seen in 242. Light sources for the growth of the algae are shown by 238 and diffusers if needed are shown by 240. FIG. 4E is an alternative embodiment of the mobile photobioreactor 300 of the present invention specifically designed if the baffles cannot mitigate possible adverse effects of acceleration and deceleration on productivity. The bioreactor 300 placed on a trailer bed or platform 314. The power supply unit 302 comprises an independent diesel generator or equivalent. The bioreactor 300 comprises a nutrient supply 304 as well as heating and cooling units 306. The transparent tubes of growing algae are seen in 312 and are placed in a vertical direction. Light sources for the growth of the algae are shown by 308 and diffusers are shown by 310.

A unique transportable algal oil extraction unit comprising a self contained and fully integrated extraction processes are described hereinabove. The mobile algal oil extraction unit of the present invention permits the oil extraction processing equipment to travel to and within the algae growth facility. The extraction unit includes harvesting (dewatering), lysing, and oil separation processes all integrated in a self contained and mobile unit. The mobile unit enables moving from pond-to-pond (or other growth mediums) without having to pipe or ship algae water over long distances. Shipping and piping large quantities of algal water may not be practical in particular when algae growth facilities are remote, distributed or too small to justify a dedicated extraction plant.

The unit may be realized in a variety of control approaches, depending on the application. The unit can be, e.g., manually controlled by an operator or controlled remotely. It may be electronically controlled by an onsite operator using appropriate sensors and either a wireless or wired control system or even controlled via satellite or other transmission whether analog or digital.

A processing challenge is the fact that the quantity of fluids processed differs greatly during processing. Typically, one hundred times the liquid is introduced into the plate settler than is passed to the lysing unit. While the lysing unit does not affect the fluid volume, the oil extracted can be 1,000 times smaller than the fluid introduced into the separation unit. This fact constrains the processing unit. It must be very cost effective, which determines the specific processing steps. It must return nearly all water to the algae growing units with appropriate chemical and biological balance. Appropriate in-line measuring equipment and processing is included in the system. Since the various steps process different quantities of a fluid stream, each step is sized for efficient use and holding tanks may be used to buffer variations in flow.

Example. Testing of a Microporous Hollow Fiber Membrane Contactor using a Propylene Gly col/Water Mixture and Terpenes.

This example details two studies that determined the capabilities of a microporous hollow fiber membrane contactor for separation of biological products. Microporous hollow fiber membrane contactors provide a large effective area for mass transfer and coalescence within a small volume. The primary objective of these tests was to operate the membrane at different compositions of water and propylene glycol on the shell side and observe any breakthrough of, in this example, terpenes on the tube side. Terpenes serve as a model for the isolation of a wide variety of hydrocarbons. The present invention can also be used to process any industrial liquid sources of oil, including but not limited non- biological sources of oil, including water that is contaminated with oil, oil that is contaminated with water, produced water, drilling mud used in oil and gas extraction, wastewater, tailings pond water, waste oil, any liquid waste or byproducts, from oil and/or gas production, transport, or processing.

Briefly, the propylene glycol (PG)/water mixture was fed on the shell-side while the terpene was fed counter-currently on the tube-side. Several trials using different concentrations of PG and water were conducted (up to 75 weight % PG) and no breakthrough across the liquid:liquid interface was observed under the operating conditions employed. These tests demonstrate that commercial mixtures can be separated using the devices and methods taught herein.

In a second study, terpene was injected into the 75%PG/25%water mixture fed to the shell-side of the membrane better simulating the future test. This trial run determined if the membrane could extract terpene from the water/PG stream. Of 44 milliliters of terpene injected in the shell-side PG/water stream, 25 milliliters were recovered in the tube-side. Since no insoluble terpene was observed in the discharge of the PG/water mixture, it is likely that the missing terpene was trapped in the membrane tube walls.

The inventors tested a microporous hollow fiber membrane contactor for breakthrough at the liquid:liquid interface as increasing concentration of propylene glycol (PG) in water is added to the shell side flow. Terpene (Orange Oil) is added to the tube side of the membrane. These results demonstrate the ability of the system to recover a wide variety of oils, e.g., orange oil, from an aqueous mixture. As the membrane showed no breakthrough, the membrane is able to efficiently separate out insoluble terpene from a water/PG and a water/ethanol stream. The membrane has the potential to selectively recover the terpene from this emulsion-like feed. The skilled artisan will recognize that the oil can coalesced against either side of the hollow fiber membrane. Further, the oil can be isolated without the addition of a solvent into the liquid source of oil. In certain embodiments, the oil can also be isolated without the need for a counterflowing fluid on the side of the membrane opposite that which is contacting the liquid source of oil.

Methods and Equipment. A microporous hollow fiber membrane contactor (e.g., a Liqui-Cel™ Membrana), was used to test breakthrough and separation. The membrane is used commercially for degassing liquids and not for the present application. The membrane resembles a heat exchanger such that it has tubes running through a shell, and a baffle to allow the shell side fluid to move radially through the membrane to enhance contact with the tubes (Dass, 2010). These tubes are actually polypropylene hollow fibers where the walls contain large pores on the order of 0.03 microns. The hollow fiber membrane surface is non-polar, making the tube side suitable for coalescing the non-polar terpene droplets. The feed solution is fed to the shell-side to minimize pressure drop while the tube side flow functions as a fluid to carry away the coalesced terpene. As a result, terpene will be fed at a slower rate on the tube side. The polarity difference is what keeps the two liquids from mixing and prevents the water and propylene glycol from flowing into the tube side of the membrane. Since the terpene preferentially wets the membrane, it is important to maintain a higher operating pressure on the non-polar side. This will allow the immobilization of a liquid-liquid interface within the pores of the fiber walls. It will be important that the bio-solids are hydrophilic so they do not adhere to the fiber surface. Also the bio-solids should be less than 40 microns to allow travel between the fibers and prevent plugging.

A full schematic of the membrane/tank system can be observed from the process flow diagram shown in FIG. 2, described hereinabove. A large open-top cone-bottomed metal tank was used to hold the water/PG mixture while a small, enclosed stainless steel tank is used to store, e.g., a terpene. A series of hoses and metal tubing connect the tanks to the membrane. Other devices may be placed in stream with the tubing and membrane contactor, e.g., mass flow meters and pressure gauges. Pumps were used to circulate the water/PG stream and the terpene stream. The aqueous pump is a Model # 33160 Moyno progressing cavity pump, which uses its rotating cavities to pull fluid forward. The terpene line pump was a TE4-HC Little Giant single stage centrifugal pump, which uses an impeller to spin the fluid out of the pump. During the separations testing, a peristaltic pump is used to inject small amounts of terpene into the system. It compresses a tube that holds the fluid in order to push the liquid forward.

Testing Procedure. A concern is that the high viscosity and lower interfacial tension associated with the propylene glycol will push the pressure beyond the membrane limit and cause breakthrough of the water/PG mixture into the terpene side of the membrane, despite the polar nature of the aqueous liquid. Therefore, this experiment looks to test this limit as well as observe less concentrated mixtures to see if breakthrough occurs with even smaller amounts of PG. Because the objective of this project is to look for polar liquid droplets in the terpene stream, the terpene tube and hose lines were blown through with nitrogen to remove any aqueous substance that could contaminate the terpene and give a false result.

The first run included pure water stream on the shell-side and a terpene stream on the tube-side.

The lack of propylene glycol is to ensure that the water is not causing breakthrough and that the membrane is fully functional. The water/PG tank is charged with 4,000 milliliters of distilled water, and the terpene feed tank is charged with 1,000 milliliters of terpene. The water stream starts circulating first to build a backpressure to prevent terpene from leaking into the shell-side of the membrane. Starting with the inlet shell-side throttle valve completely open, the valve is slowly closed to increase the pressure drop to a suitable backpressure. Once a steady flow of the water stream is established, the terpene stream pump is turned on to allow flow through the tubes and membrane. Then, after recording data from the pressure gauges and flow meters, the system runs for thirty minutes. After the allotted time, the terpene pump is turned off first to maintain backpressure. The terpene is drained from the system to observe if insoluble droplets appear and to record any volume loss. The water stream is constantly running through these trials.

In a second run, the water stream included 25 weight percent (weight %) of propylene glycol (PG). The 25 weight percent (wt%) equals about 1,250 milliliters of PG, for a total tank volume of 5250 milliliters. The shell-side stream is allowed to circulate for five minutes to mix the PG and water together. A fresh 1,000 milliliters of terpene is added to the tube-side tank to replace the drained terpene from the first run. Once the water/PG stream is well mixed, the terpene pump is turned on and the system is allowed to run for thirty additional minutes. After recording the data from the flow meters and pressure gauges, the terpene pump is turned off. The tube-side line is drained and the terpene is again analyzed for the presence of insoluble droplets and volume changes.

In a third run, the PG content was increased in the shell-side stream to 50 weight %. In other words, an additional 2,600 milliliters of PG is added for a total shell-side tank volume of 7,850 milliliters. When propylene glycol is added, the density of the liquid increases. With the same flow rate, the amount of material being pumped through the tubes and membrane increases, which increases the pressure drop at the liquid:liquid interface. The throttle valve is opened slightly to prevent the increase in pressure from blowing out the membrane, while still maintaining a high enough backpressure. The shell-side stream is again allowed to circulate for five minutes to mix, while a fresh 1,000 milliliters of terpene is added to replace the drained terpene from the second run. The terpene pump is turned on, and the system runs for thirty minutes. After the pertinent data is recorded, the terpene line is turned off and drained. The drained oil is then analyzed for the presence of insoluble droplets and volume changes.

Another run used a propylene glycol (PG) content to the maximum 75 weight percent for these tests. This equals an additional 7,650 milliliters of PG for a total tank volume of 15,500 milliliters. During the five minutes of mixing, an additional 1,000 milliliters of fresh terpene is added to the tube-side tank. The terpene pump is turned on and the system is left to run. The appropriate pressure gauge and flow meter measurements are recorded. After thirty minutes, the terpene pump was turned off and the tube- side line is drained. The terpene phase is visually analyzed a final time for insoluble droplets and volume loss.

Once the final charge of terpene was drained, the back-pressure was no longer needed and the water/PG stream pump can be turned off. If no insoluble droplets are found in the terpene in any of the trial runs, this experiment proves that breakthrough is not observed with these aqueous and collection fluid combinations. If breakthrough is observed, we will identify an upper limit of water/PG mix that can be tolerated by the system.

The testing set-up is very similar to the initial testing procedure. The original 75 weight-percent PG in water mixture is in the shell-side tank. This stream is circulated first to set up the appropriate back pressure. A fresh 1,000 milliliters of terpene is carefully measured into the tube-side tank and then circulated through the system. An initial 25 milliliters of terpene is injected using a peristaltic pump into the water/PG stream. The peristaltic pump rate is set at 0.4 milliliters per minute and the system is left to run for two hours. Periodically, additional terpene is added to the water/PG stream to keep the flow constant. After two hours, the peristaltic pump is turned off and the final volume of terpene injected into the polar fluid stream is recorded. The circulation of the pure terpene stream on the tube-side of the membrane is stopped and drained. Using a volumetric flask and a graduated cylinder, the total volume of terpene is recorded to determine if there is more terpene in the tube-side tank than the initial 1,000 milliliters. If an increase of volume is observed, then the membrane is effective at removing the injected terpene from the water/PG stream. Analysis can be done to determine how efficiently the membrane separates the two streams.

Testing Results. The results of the breakthrough testing with terpene and water/PG showed no evidence of breakthrough, even at the highest PG/water ratio. Despite the increase in density in the water/PG stream as more PG was added, no breakthrough could be seen in either stream. The water/PG stream smelled slightly like terpene, although no droplets or beads of oil were seen on the surface or walls of the shell-side tank. While the solubility of terpene in water is low, a very small amount of the terpene that contacts the water at the liquid-liquid barrier in the membrane may dissolve. It is known that the odor thresholds for terpenes range from between 0.1 to 1.7 ppm, which means that it is possible to smell the small amount of terpene that dissolves in the stream. This data is significant because it explains that smelling the terpene in the shell-side tank does not necessarily mean breakthrough, just a minimal amount of solubility. The testing data for each trial run can be found in Table 4, and design parameters can be found in Table 5. The flow rate data in Table 1 shows that the shell-side outlet flow was consistently about 4 to 5 lbs./hr. higher than the shell-side inlet flow. They should have been almost the same, indicating that one of the meters may not have been calibrated properly. An indicator that breakthrough occurred would be a lower outlet flow rate due to a loss of liquid in the water/PG stream, but that is not the case here. Also, Table 1 shows a dramatic increase in mass flow rate as the PG concentration increases, even though the backpressure and pump frequency are kept around relatively the same number. The high PG density means more mass is moving through the flow meter.

Table 4 - Recorded Data from Trials 1 through 4.

Table 5 - Fluid Data.

The first trial run used 4,000 milliliters of distilled water on the shell-side, and 1 ,000 milliliters of terpene on the tube-side. No water was found on the surface of the terpene after draining, which indicates that breakthrough did not occur. However, around 300 milliliters of terpene was unaccounted for. The terpene stream had difficulty initializing a steady flow because air bubbles prevented the tube- side pump from priming. Venting the stream resulted in leaks and spills, which contributed to the liquid loss, but was effective at removing air bubbles and priming the pump. Also, because the tube-side lines were nitrogen-blown to remove any excess water, this initial run lost more liquid as a certain amount of terpene became lodged in the walls of the pipes and membrane. When drained, the initially clear terpene took on a cloudy orange color. It is speculated that this color originates from old canola oil that was trapped in the apparatus' piping from previous studies.

The second trial run used 4,000 milliliters of distilled water, 1,250 milliliters of propylene glycol (PG), and 1,000 milliliters of fresh terpene. At the end of the run, no water or insoluble liquid was found on the surface of the terpene, indicating no breakthrough. Also, there was no loss of volume of the terpene. More caution was used during this run when venting the lines to prime the pump, and any spilled liquid was captured and returned to the terpene tank. No terpene became trapped on the walls of the system either because a small amount still remained from the first trial run. The terpene still contained an orange color after the trial; however, the liquid was transparent.

The third trial run used 4,000 milliliters of distilled water, 3,850 milliliters of PG and 1,000 milliliters of fresh terpene. No insoluble liquid was found in the terpene after it was drained, indicating no breakthrough. A small amount of terpene, roughly 50 milliliters was lost in the run, which is possibly due to a poor drain of the tube-side lines. The terpene continued to show an orange color after running through the system, although it was a lighter orange than the previous runs.

In the final run 4,000 milliliters of distilled water was used along with 11,500 milliliters of PG, and

1,000 milliliters of terpene. No insoluble liquid was found in the terpene after draining, demonstrating that the maximum 75 weight% mixture of propylene glycol and water still does not cause breakthrough. Similar to the second trial, no terpene was lost in this run. The drained terpene still sustained a light orange color similar to that of the third trial run.

Separation Testing Results. The objective of the separation testing was to determine if the Liqui-

Cel membrane contactor would extract terpene from the water/PG stream. With success, the next step was to determine the membrane's efficiency. The testing data recorded from the separation trial can be seen in Table 6, and the design parameters in Table 7.

Table 6 - Recorded Data from Separations Testing.

Shell-Side Outlet 5.1 5.3 5.3

Pressure (PSIG)

Shell-Side Inlet Flow 417.0 ± 3 440.0 ± 2 440.0 ± 5

(lb/hr)

Tube- Side Inlet Flow 19.00 ± 3 14.00 ± 2 15.00 ± 4

(lb/hr)

Shell-Side Outlet Flow 424.0 ± 3 443.0 ± 2 445.0 ± 4

(lb/hr)

7 - Separations Fluid Data.

Duration (min) 120

Shell Side (ml) [3:1 Water/Propylene Glycol] 15,500 Tube Side (ml) [Terpene] 1,000 Terpene Injected (ml) 44

Terpene Excess (ml) 25

Efficiency (%) 56

Peristaltic Pump Rate (ml/min) 0.4

A total of 44 milliliters of terpene was injected into the water/PG stream via a burette-fed peristaltic pump running at 0.4 milliliters per minute. Exactly 1,000 milliliters of terpene was measured into the tube-side tank. After two hours the terpene line was stopped and drained, and a measurement was taken of the volume of liquid. An additional 25 milliliters of terpene made its way into the tube-side lines, proving that the membrane does have the capability of separating the terpene out of the water/PG stream.

The initial tests provide evidence that the breakthrough is unlikely. As an added test, terpene was metered into the re-circulating 75% PG/25% water mixture. In this case, a known amount of terpene was injected into the water/PG stream via a peristaltic pump. The objective was to determine if the non-polar terpene can be separated out of the polar water/PG stream using the membrane contactor under these operating parameters. The process flow diagram can be seen in Figure 2.

It was found that the membrane is truly capable of large scale, consistent separation of oil because at these small scales some terpene inevitably gets trapped in the membrane and pipe walls. By simply recovering larger amounts of terpene will minimize the inherent losses in the tube walls. The testing was successful in regards to observing that breakthrough never occurred, even at 75 weight % propylene glycol in water.

The separation was successful for removing terpene from the water/PG stream. Now, the system just needs to run in a more commercially relevant manner. For the next testing, a larger amount of terpene should be injected into the water/PG stream. This way, any terpene that gets trapped in the walls or membrane will be a smaller fraction of the total amount. Also, increasing the run time to half a day or a full day will give any extra terpene a chance to fully flow through the system multiple times. This way, more terpene is exposed to the membrane and will have more of a chance to separate.

Calculations. Converting 25 Weight% PG to Relative Volume of PG.

m _w = mass of water (g)

fpc ⁼ weight fraction of PG in water

p _PG = density of PG (g/ml)

VPG = volume of PG (ml)

4000m/ H ₂ 0 = 4000# H ₂ 0

4000 g H ₂ 0

= 5333.3 q Total

0.75 ^y

5333.3g Total - 4000# H ₂ 0 = 1333.

1333.3g PG

1287ml PG ~ 1250ml PG

1.036 S PG

ml

Determining the Separation Efficiency of the Membrane. 100 = Eff.

(2)

V _E = volume of extracted terpene (ml)

Vi = volume of initial terpene (ml)

Eff. = percent efficiency of membrane

25

— x lOO = 56.82

44

The present invention can be summarized in a series of flowcharts (FIGS. 5-10) that show the various embodiments of the present invention. FIG. 5 shows one basic flowchart of the present invention in which a feed or liquid source 10 of oils is processed in the steps as outlined. In FIG. 5, the feed or liquid source 10 is then concentrated, harvested and/or dewatered 12 by one or more of the processes and/or methods taught hereinabove. In certain non-limiting examples the fees source is a biological source of oils, but could also be hydrocarbons that are mixed in with one or more contaminants or aqueous components. For biological sources of oils, the biological cells (e.g., yeast, plants, algae, bacteria, insect, etc.) are lysed at step 14 using one or more of the non-limiting examples of processes and/or methods taught hereinabove. Next, the one or more oils from the feed or liquid source 10 are recovered at step 16 by coalesce on the surface of the one or more membrane contactors taught hereinabove. In this example, the biomass or remainder of the feed that is not coalesced can be further processed at step 18 into fertilizer, animal feed, or additional feedstock for energy sources. The coalesced oil can then be further processed at step 20 into one or more products.

FIG. 6 shows a process in which the feed or liquid source 10 is then lysed at step 14 using one or more of the non-limiting examples of processes and/or methods taught hereinabove. Next, the one or more oils from the feed or liquid source 10 is recovered at step 16 by coalesce on the surface of the one or more membrane contactors taught hereinabove. In this example, the biomass or remainder of the feed that is not coalesced can be further processed at step 18 into fertilizer, animal feed, or additional feedstock for energy sources. The coalesced oil can then be further processed at step 20 into one or more products.

FIG. 7 shows another basic flowchart of the present invention in which a feed or liquid source 10 of oils is recovered at step 16 by coalescence on the surface of the one or more membrane contactors taught hereinabove. In this example, the biomass or remainder of the feed that is not coalesced can be further processed at step 18 into fertilizer, animal feed, or additional feedstock for energy sources or fed back into the feed or liquid source 10 for additional growth or recovery of oils at step 16. The coalesced oil can then be further processed at step 20 into one or more products.

In FIG. 8, the feed or liquid source 10 is then concentrated, harvested and/or dewatered 12 by one or more of the processes and/or methods taught hereinabove. Next, the one or more oils from the feed or liquid source 10 are recovered at step 16 by coalesce on the surface of the one or more membrane contactors taught hereinabove. In this example, the biomass or remainder of the feed that is not coalesced can be further processed at step 18 into fertilizer, animal feed, or additional feedstock for energy sources. The coalesced oil can then be further processed at step 20 into one or more products.

In FIG. 9 a feed or liquid source 10 of soluble or insoluble oils (and optionally lysed at step 14) using one or more of the non-limiting examples of processes and/or methods taught hereinabove, if the source of oil is biological. Next, the one or more oils from the feed or liquid source 10 are recovered at step 16 by coalesce on the surface of the one or more membrane contactors taught hereinabove. In this optional example, is the source of oil is biological, then the biomass or remainder of the feed that is not coalesced can be further processed at step 18 into fertilizer, animal feed, or additional feedstock for energy sources. The coalesced oil can then be further processed at step 20 into one or more products.

In FIG. 10, the feed or liquid source 10 of soluble or insoluble oils is then concentrated, dewatered (or if harvested from a biological source) 12 by one or more of the processes and/or methods taught hereinabove, without the addition of a solvent. In certain non-limiting examples the feed sources, such as biological or non-biological sources of oil, but could also be hydrocarbons that are mixed in with one or more contaminants or aqueous components. If the source of soluble or insoluble oils is biological, the biological cells (e.g., yeast, plants, algae, bacteria, insect, animal, cyanobacteria, etc.) are lysed at optional step 14 using one or more of the non-limiting examples of processes and/or methods taught hereinabove. Next, the one or more oils from the feed or liquid source 10 are recovered at step 16 by coalesce on the surface of the one or more membrane contactors taught hereinabove. In this example, the biomass or remainder of the feed that is not coalesced can be further processed at step 18 into fertilizer, animal feed, or additional feedstock for energy sources and/or can be reprocessed back into the oil recovery membrane to continue to capture oil. The coalesced oil can then be further processed at step 20 into one or more products.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value or the variation that exists among the study subjects.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The term "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.