Provided are biomass-based materials and valuable uses of microalgal biomass including: (i) acetylation of microalgal biomass to produce a material useful in the production of thermoplastics; (ii) use of triglyceride containing microalgal biomass for production of thermoplastics; (iii) combination of microalgal biomass and at least one type of plant polymer to produce a material useful in the production of thermoplastics; (iv) anionization of microalgal biomass to form a water absorbant material; (v) cationization of microalgal biomass, and optional flocculation, to form a water absorbant material; (vi) crosslinking of anionized microalgal biomass; (vii) carbonization of microalgal biomass; and (viii) use of microalgal biomass in the making of paper.
Claims

1. A thermoplastic composition or thermoset composition comprising one or more of a covalently modified microbial biomass from an oleaginous microbe and a non-covalently modified biomass from a heterotrophically cultivated microbe, wherein the microbial biomass optionally comprises from 0.25% to 90% triglyceride by dry cell weight; the thermoplastic composition optionally further comprising one or more plant polymers.

2. The composition according to claim 1, wherein the microbe is an oleaginous microbe.

3. The composition according to claim 1, wherein the microbe has been lysed.

4. The composition according to claim 1, wherein the biomass is microalgal biomass.

5. The composition according to claim 4, wherein the microalgal biomass is derived from cells having a mean diameter of between 1 micron and 50 microns.

6. The composition according to claim 4, wherein the microalgal biomass comprises from 0.25% to 20% triglyceride by dry cell weight.

7. The composition according to claim 1, further comprising one or more plant polymers.

8. The composition according to claim 4, wherein the covalently modified microalgal biomass has been covalently modified with a hydrophobic group, a hydrophilic group, an anionic group or a cationic group.

9. The composition of claim 8, wherein the covalently modified microalgal biomass is microalgal biomass that has been modified by one or more reactions selected from the group consisting of acylation, hydroxylation, epoxidation, isocyanization, and silylation.

10. The composition of claim 9, wherein the acylation reaction is acetylation.

11. The composition of claim 8, wherein polysaccharide of the microalgal biomass is covalently modified.

12. The composition according to claim 9, wherein the covalently modified algal biomass is characterized by a DS value of 0.25 to 3.

13. The composition according to claim 4, wherein the microalgal biomass is unbleached.

14. The composition according to claim 4, wherein the microalgal biomass comprises less than 5000 ppm color generating compounds.

15. The composition according to claim 4, wherein the microalgal biomass comprises less than 3000 ppm chlorophyll.

16. The composition according to claim 4, wherein the biomass is of microalgae that are heterotrophs, and optionally obligate heterotrophs.

17. The composition according to claim 4, wherein the microalgae are of the class Trebouxiophyceae.

18. The composition according to claim 17, wherein the microalgae are of the genus Chlorella or the genus Prototheca.

19. The composition according to claim 18, wherein the microalgae are Prototheca moriformis.

20. The composition according to claim 4, wherein the thermoplastic composition further comprises a plasticizer.

21. The composition according to claim 20, wherein the plasticizer is selected from a group consisting of one or more of: glycerol, sorbitol, triacetin, triethyl citrate, acetyl triethyl citrate, tributyl cirtate, acetyl tributyl citrate, trioctyl citrate, acetyl trioctyl citrate, trihexyl citrate, butyryl trihexyl citrate, trimethyl citrate, alkyl sulphonic acid phenyl ester, and 1,2-cyclohexane dicarboxylic acid diisononyl ester.

22. The composition according to claim 1, wherein the composition further comprises a surfactant.

23. The composition according to claim 22, wherein the surfactant is selected from the group consisting of glyceryl monostearate, ethoxylated dimethylsiloxane, polyoxyethylene, propylene oxide, an organic sulfate, an organic sulfonate, an alkyl polyglycoside, and a polyolefin glycol.

24. A blended composition comprising a thermoplastic composition according to claim 1, and a second thermoplastic composition.

25-27. (canceled)

28. The composition according to claim 1, wherein the thermoplastic composition has one or more of the following characteristics: (a) a Young's modulus of 300-3000 MPa; (b) a tensile strength of 5-70 MPa; (c) a tensile strength at maximum load of 5-50 MPa; or (d) an ultimate elongation of 1-400%.

29. The composition according to claim 1, wherein the fatty acid profile of the triglyceride comprises at least 60% C18:1; at least 50% combined total amount of C10, C12, and C14; or at least 70% combined total amount of C16:0 and C18:1.

30. The composition according to claim 1, wherein the microbial biomass is a fraction that is insoluble in an aqueous solvent, said insoluble fraction produced by removing components soluble in an aqueous solvent from microbial biomass.

31. The composition according to claim 1, wherein the composition has been formed through extruding, molding, blowing, coating, calendering, or spinning.

32. The thermoplastic composition according to claim 14, wherein the composition is a film or a fiber.

33. The thermoplastic composition according to claim 1, wherein the one or more plant polymers is from the group consisting of switchgrass, rice straw, sugar beet pulp, corn starch, potato starch, cassaya starch, sugar cane bagasse, soybean hulls, dry rosemary, cellulose, corn stover, delipidated cake from soybean, canola, cottonseed, sunflower, jatropha seeds, paper pulp, and waste paper.

34. An absorbent composition comprising microbial biomass from a microbe covalently modified with a hydrophilic moiety and wherein the composition is optionally cross-linked.

35-69. (canceled)

70. A method of making an adsorbent material, wherein the method comprises the steps of: a) preparing biomass from a microbe; b) hydrothermally carbonizing the biomass, thereby making the adsorbent material.

71-86. (canceled)

87. A paper product comprising 0.1% to 50% biomass from heterotrophic ally cultivated microbes.

88-106. (canceled)

107. A method of making a thermoplastic composition or a thermoset composition, the method comprising the steps of: a) providing biomass from heterotrophically cultivated microbes; b) acylating the polysaccharides within the biomass, wherein the acylating is optionally acetylating; c) adding one or more of a plasticizer, an additional polymer, a filler, or a cross-linking agent d) optionally adding one or more plant polymers.

108-124. (canceled)

125. A process for producing triglyceride comprising (a) heterotrophically cultivating microalgal cells in a culture medium comprising crop-derived sugar so as to produce triglyceride inside the cells; (b) removing the triglyceride from the cells to produce an oil and a residual biomass; (c) hydrothermally carbonizing a water soluble fraction and/or water insoluble fraction of the biomass to produce a carbonized product and a nutrient-rich aqueous solution; and (d) repeating the process with recycling of the nutrients of the nutrient-rich aqueous solution to step (a) to support the cultivation of additional microalgal cells or using the nutrients of the nutrient-rich aqueous solution in the growing of crops.

126-133. (canceled)

134. A composition comprising a blend of a moldable polymer, a microalgal biomass, and optionally a lipid selected from a triacylglyceride, a fatty acid, a fatty acid salt, a fatty acid ester, and one or more combinations thereof, wherein the microalgal biomass is optionally covalently modified and is obtained from a heterotrophic oleaginous microalgae.

135-176. (canceled)

177. A film comprising a composition of claim 134.

178. An injection molded article comprising a composition of claim 134. Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 61/579,961, filed Dec. 23, 2011, U.S. Provisional Patent Application No. 61/615,832, filed Mar. 26, 2012, U.S. Provisional Patent Application No. 61/616,356, filed Mar. 27, 2012, U.S. Provisional Patent Application No. 61/671,066, filed Jul. 12, 2012, U.S. Provisional Patent Application No. 61/691,210, filed Aug. 20, 2012, U.S. Provisional Patent Application No. 61/701,530, filed Sep. 14, 2012, and U.S. Provisional Patent Application No. 61/728,807, filed Nov. 21, 2012. Each of these applications is incorporated herein by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] The present invention relates to materials produced using biomass that include cell wall remains of heterotrophically cultivated single cells. In particular, the biomass can be used to produce products including plastic, paper, adsorbent, or absorbant materials.

BACKGROUND

[0003] Algae, and especially microalgae (single celled algae) have been the subject of recent interest in terms of the production of lipids and fatty acids for use in fuels, chemicals, soaps, and foods. As disclosed in WO2008/151149 and WO2010/063032, certain species of microalgae can be cultured on a fixed carbon source (e.g., glucose, sucrose, glycerol or hydrolyzed cellulosic material) without the use of sunlight to produce high yields of lipid as measured as a percentage of dry cell weight. Some species of miroalgae are obligate heterotrophs; they lack the ability to use sunlight and so must grow on a fixed carbon source (i.e., not carbon dioxide). The aforementioned patent applications also teach that microalgae can be genetically engineered to allow growth on sucrose and to alter the chain length and saturation profiles of the fatty acids produced by the microalgae. Thus, the microalgae can be used as a biocatalyst to upconvert sugar into more valuable products. Other technologies use autotrophic algae, bacteria, yeast or cyanobacteria to produce oil from sugar.

SUMMARY

[0004] In one aspect, the invention provides thermoplastic compositions or thermoset compositions. In some embodiments, the thermoplastic compositions or thermoset compositions comprise one or more of a covalently modified microbial biomass from an oleaginous microbe and a non-covalently modified biomass from a heterotrophically cultivated microbe, wherein the microbial biomass optionally comprises from 0.25% to 90% triglyceride by dry cell weight. In some embodiments, the microalgal biomass comprises from 0.25% to 20% triglyceride by dry cell weight. In some embodiments, the fatty acid profile of the triglyceride comprises at least 60% C18:1; at least 50% combined total amount of C10, C12, and C14; or at least 70% combined total amount of C16:0 and C18:1. The thermoplastic composition may optionally further comprise one or more plant polymers. Suitable plant polymers include, e.g., switchgrass, rice straw, sugar beet pulp, corn starch, potato starch, cassaya starch, sugar cane bagasse, soybean hulls, dry rosemary, cellulose, corn stover, delipidated cake from soybean, canola, cottonseed, sunflower, jatropha seeds, paper pulp, and waste paper. In various embodiments, the microbe is an oleaginous microbe. In some embodiments, the microbe has been lysed. In some embodiments, the biomass is microalgal biomass. In some embodiments, the microalgal biomass is derived from cells having a mean diameter of between 1 micron and 50 microns. In various embodiments, the microalgal biomass comprises one or more plant polymers. In some embodiments, the covalently modified microalgal biomass has been covalently modified with a hydrophobic group, a hydrophilic group, an anionic group or a cationic group. In some embodiments, the covalently modified microalgal biomass is microalgal biomass that has been modified by one or more reactions selected from the group consisting of acylation, hydroxylation, epoxidation, isocyanization, and silylation. In a particular embodiment, the acylation reaction is acetylation. In some embodiments, polysaccharide of the microalgal biomass is covalently modified. In some embodiment, the covalently modified algal biomass is characterized by a degree of substitution ("DS") value in the range of 0.25 to 3. In some embodiments, the microalgal biomass is unbleached. In various embodiments, the microalgal biomass comprises less than 5000 ppm color generating compounds (e.g., chlorophyll). In various embodiments, the microalgal biomass comprises less than 3000 ppm chlorophyll. In some embodiments, the biomass is of microalgae that are heterotrophs, and optionally obligate heterotrophs. In some embodiments, the microalgae are of the class Trebouxiophyceae. In some embodiments, the microalgae are of the genus Chlorella or the genus Prototheca. In a particular embodiment, the microalgae are Prototheca moriformis. In some embodiments, the thermoplastic composition further comprises a plasticizer. Suitable plasticizers include, e.g., glycerol, sorbitol, triacetin, triethyl citrate, acetyl triethyl citrate, tributyl cirtate, acetyl tributyl citrate, trioctyl citrate, acetyl trioctyl citrate, trihexyl citrate, butyryl trihexyl citrate, trimethyl citrate, alkyl sulphonic acid phenyl ester, and 1,2-cyclohexane dicarboxylic acid diisononyl ester. In some embodiments, the composition further comprises a surfactant. Suitable surfactants include, e.g., glyceryl monostearate, ethoxylated dimethylsiloxane, polyoxyethylene, propylene oxide, an organic sulfate, an organic sulfonate, an alkyl polyglycoside, and a polyolefin glycol. In various embodiments, the microbial biomass is a fraction that is insoluble in an aqueous solvent, said insoluble fraction produced by removing components soluble in an aqueous solvent from microbial biomass. In various embodiments, the microbial biomass is insoluble in an aqueous solvent. In various embodiments, the composition has been formed through extruding, molding, blowing, coating, or calendering. In various embodiments, the composition is a film.

[0005] In a further aspect, the invention provides blended compositions. In various embodiments, the blended compositions comprise a thermoplastic composition as described above and herein, and a second thermoplastic composition. In some embodiments, the second thermoplastic composition is present in the range of 5 to 95% by mass. Suitable second thermoplastic compositions include, e.g., polylactic acid, polycaprolactone, polyesteramide, polyhydroxybutyrate, polyhydroxybutyrate-co-valerate, polyhydroxyalkanoate, polyethylene, polypropylene, polyethylene terephthalate, and polycarbonate. In some embodiments, the second thermoplastic composition is a derivative of polyethylene. In some embodiments, the second thermoplastic composition is a derivative of polypropylene. In some embodiments, the second thermoplastic composition is of biological origin. In some embodiments, the thermoplastic composition has one or more of the following characteristics:

[0006] (a) a Young's modulus of 300-3000 MPa;

[0007] (b) a tensile strength of 5-70 MPa;

[0008] (c) a tensile strength at maximum load of 5-50 MPa; and/or

[0009] (d) an ultimate elongation of 1-400%.

[0010] In a related aspect, the invention provides absorbent compositions. In various embodiments, the absorbent compositions comprise thermoplastic compositions or thermoset compositions as described above and herein. In various embodiments, the absorbent compositions comprise microbial biomass from a microbe covalently modified with a hydrophilic moiety. In some embodiments, the absorbent composition is cross-linked. In various embodiments, the microbe is an oleaginous microbe. In some embodiments, the microbe has been lysed. In some embodiments, the microbe is a microalga. In some embodiments, the microalga cell has a mean diameter of between approximately 1 micron and approximately 50 microns. In various embodiments, the hydrophilic moiety is anionic, cationic, zwitterionic, or neutral. In some embodiments, the anionic moiety is a carboxylate, a sulfate, a sulfonate, or a phosphate. In some embodiments, the cationic moiety is an amine or a substituted amine. In some embodiments, the neutral moiety is an hydroxyl or acyl. In a particular embodiment, the anionic group is a carboxylate group, and the covalently modified biomass is formed by modifying the biomass with a carboxymethyl group. In some embodiments, the modified biomass is characterized by a degree of substitution ("DS") value of 0.25 to 3. In some embodiments, the covalently modified biomass comprises polysaccharide. In some embodiments, the absorbent compositions further comprise a cross-linking agent. Suitable cross-linking agents include, e.g., aldehydes, C2-C8 dialdehydes, C2-C9 polycarboxylic acids, epichlorhydrin, divinyl sulphone, ethylenediamine, cystamine dihydrochloride, acrylic acid, sorbitan monolaurate, polyethylene glycol, sodium zirconium lactate, sodium borate, genipin, and sodium stearate. In a particular embodiment, the dialdehyde is glyoxal. In various embodiments, the absorbent composition is included in a structural material. In some embodiments, the fatty acid composition of the microbial biomass comprises at least 60% C18:1; at least 50% combined total amount of C10, C12, and C14; or at least 70% combined total amount of C16:0 and C18:1. In some embodiments, the microbial biomass is a biomass fraction that is insoluble in an aqueous solvent, said insoluble fraction produced by removing components soluble in an aqueous solvent from microbial biomass. In some embodiments, the microbial biomass is insoluble in an aqueous solvent. In various embodiments, the composition absorbs at least 5 times its weight in liquid. In some embodiments, the composition absorbs at least 5 times its weight in liquid after immersion in liquid for 4 hrs. In various embodiments, the composition absorbs at least 10 times its weight in liquid. In some embodiments, the composition absorbs at least 10 times its weight in liquid after immersion in liquid for 4 hrs. In various embodiments, the composition absorbs at least 20 times its weight in liquid. In some embodiments, the composition absorbs at least 20 times its weight in liquid after immersion in liquid for 4 hrs. In various embodiments, the composition absorbs at least 50 times its weight in liquid. In some embodiments, the composition absorbs at least 50 times its weight in liquid after immersion in liquid for 4 hrs. In various embodiments, the composition absorbs at least 100 times its weight in liquid. In some embodiments, the composition absorbs at least 100 times its weight in liquid after immersion in liquid for 4 hrs. In various embodiments, the liquid is water, saline, oil, urine, or blood. In some embodiments, the biomass is of microalgae that are heterotrophs, and optionally obligate heterotrophs. In some embodiments, the microalgae are of the class Trebouxiophyceae. In some embodiments, the microalgae are of the genus Chlorella or the genus Prototheca. In a particular embodiment, the microalgae are Prototheca moriformis. In various embodiments, the absorbent composition further comprises a plant polymer. Suitable plant polymers include, e.g., switchgrass, rice straw, sugar beet pulp, sugar cane bagasse, soybean hulls, corn starch, potato starch, cassaya starch, dry rosemary, cellulose, corn stover, delipidated cake from soybean, canola, cottonseed, sunflower, jatropha seeds, paper pulp, and waste paper. In some embodiments, the composition further comprises a second absorbent composition. Suitable second absorbent compositions include, e.g., polyacrylate, polyacrylamide, polyvinyl alcohol, starch, starch-g-polyacrylonitrile, cellulose, carboxymethyl cellulose, and hydroxyethyl cellulose.

[0011] In another aspect, the invention provides methods of making an adsorbent material, wherein the method comprises the steps of: a) preparing biomass from a microbe; and b) hydrothermally carbonizing the biomass, thereby making the adsorbent material. In various embodiments, the microbe is an oleaginous microbe. In some embodiments, the microbe has been lysed. In some embodiments, the microbe is microalga. In some embodiments, the microalgal biomass is prepared from microalgal cells having a mean diameter between approximately 1 micron and approximately 50 microns. In some embodiments, the biomass is of microalgae that are heterotrophs, and optionally obligate heterotrophs. In some embodiments, the microalgae are of the class Trebouxiophyceae. In some embodiments, the microalgae are of the genus Chlorella or the genus Prototheca. In some embodiments, the microalgae are Prototheca moriformis. In some embodiments, microalgal biomass is carbonized in the presence of an acidic catalyst. In various embodiments, the amount of acidic catalyst is in the range of 0.01 grams to 0.6 grams per gram of microalgal biomass. In various embodiments, the microalgal biomass is hydrothermally carbonized by heating to between about 180.degree. C. to 350.degree. C. in the presence of water from 60 minutes to 180 minutes. In some embodiments, the fatty acid composition of the biomass comprises at least 60% C18:1; at least 50% combined total amount of C10, C12, and C14; or at least 70% combined total amount of C16:0 and C18:1. In some embodiments, the biomass is a biomass fraction that is insoluble in an aqueous solvent, said insoluble fraction produced by removing components soluble in an aqueous solvent from oleaginous microbial biomass. In some embodiments, the adsorbent material further comprises a plant polymer. Suitable plant polymers include, e.g., switchgrass, rice straw, sugar beet pulp, sugar cane bagasse, soybean hulls, dry rosemary, corn starch, potato starch, cassaya starch, cellulose, corn stover, delipidated cake from soybean, canola, cottonseed, sunflower, jatropha seeds, paper pulp, and waste paper. In some embodiments, the methods further comprise the step of recovering and optionally using one or more nutrient from the biomass. Suitable nutrients include, e.g., phosphorus, nitrogen, and potassium. In various embodiments using is recycling the one or more nutrient to support the cultivation of additional microbial cells or using the one or more nutrient as a fertilizer to support plant growth.

[0012] In a related aspect, the invention provides paper products. In various embodiments, the paper products comprise thermoplastic compositions or thermoset compositions as described above and herein. In various embodiments, the paper products comprise 0.1% to 50% biomass from heterotrophically cultivated microbes. In some embodiments, the microbe is an oleaginous microbe. In some embodiments, the microbe has been lysed. In some embodiments, the microbe is a microalga. In some embodiments, the microalgal biomass is derived from microalgal cells having a mean diameter between approximately 1 micron and approximately 50 microns. In some embodiments, the biomass is of microalgae that are obligate heterotrophs. In some embodiments, the microalgae are of the class Trebouxiophyceae. In some embodiments, the microalgae are of the genus Chlorella or the genus Prototheca. In a particular embodiment, the microalgae are Prototheca moriformis. In some embodiments, the biomass is a biomass fraction that is insoluble in an aqueous solvent, said insoluble fraction produced by removing components soluble in an aqueous solvent from microalgal biomass. In some embodiments, the biomass is insoluble in an aqueous solvent. In various embodiments, the biomass is a biomass fraction that is insoluble in an aqueous solvent, said insoluble fraction produced by removing components soluble in an aqueous solvent from oleaginous microbial biomass. In some embodiments, triglyceride has been removed from the microalgal cells. For example, in various embodiments, the amount of triglyceride removed from the cells is more than 10% of the dry weight of the microalgal cells. In some embodiments, one or more cationic retention aids have been added to the biomass. Suitable cationic retention aids include, e.g., polydiallyldimethylammonium chlorides, branched polyacrylamides, polyamines having a molar mass of more than 50,000, modified polyamines grafted with ethylenimine, crosslinked polyetheramides, polyvinylimidazoles, polyvinylpyrrolidines, polyvinylimidazolines, polyvinyltetrahydropyrines, poly(dialkylaminoalkylvinylethers), poly(diakylaminoalkyl(meth)acrylates) in protonated or quaternized form, polyamidoamines obtained from a dicarboxylic acid, polyalkylenepolymines grafted with ethylenimine and crosslinked with polyethylene glycol dichlorohydrin ether, polyamidoamines reacted with epichlorohydrin to give water-soluble condensates, cationic starches, alum, polyaluminum chloride, and combinations thereof. In various embodiments, the paper products further comprise a flocculating agent. In various embodiments, the fatty acid composition of biomass comprises at least 60% C18:1; at least 50% combined total amount of C10, C12, and C14; or at least 70% combined total amount of C16:0 and C18:1. In various embodiments, one or more additional papermaking fiber has been added to the biomass. Suitable papermaking fibers include, e.g., cotton, straw, flax, jute hemp, bagasse, eucalyptus, maple, birch, aspen, pine, bamboo, rayon, polyester, fibers from recycled paper products and mixtures thereof. In some embodiments, the paper product further comprises a plant polymer. Suitable plant polymers include, e.g., switchgrass, rice straw, sugar beet pulp, sugar cane bagasse, soybean hulls, dry rosemary, corn starch, potato starch, cassaya starch, cellulose, corn stover, delipidated cake from soybean, canola, cottonseed, sunflower, jatropha seeds, paper pulp, and waste paper.

[0013] In another aspect, the invention provides methods of making a thermoplastic composition or a thermoset composition. In some embodiments, methods comprise the steps of: a) providing biomass from heterotrophically cultivated microbes; b) acylating the polysaccharides within the biomass, wherein the acylating is optionally acetylating; c) adding one or more of a plasticizer, an additional polymer, a filler, or a cross-linking agent. In various embodiments, the methods further comprise the step d) adding one or more plant polymers. Suitable plant polymers include, e.g., switchgrass, rice straw, sugar beet pulp, sugar cane bagasse, soybean hulls, dry rosemary, corn starch, potato starch, cassaya starch, cellulose, corn stover, delipidated cake from soybean, canola, cottonseed, sunflower, jatropha seeds, paper pulp, and waste paper. In some embodiments, the microbe is an oleaginous microbe. In some embodiments, the microbe has been lysed. In some embodiments, the acylating comprises acetylating using acetic anhydride or acetyl chloride as an acetylating agent. In some embodiments, the additional polymer is biodegradable. In some embodiments, the microbe is a microalga. In some embodiments, microalgal biomass is derived from microalgal cells having a mean diameter between approximately 1 micron and approximately 50 microns. In some embodiments, the biomass is of microalgae that are heterotrophs, and optionally obligate heterotrophs. In some embodiments, the microalgae are of the class Trebouxiophyceae. In some embodiments, the microalgae are of the genus Chlorella or the genus Prototheca. In a particular embodiment, the microalgae are Prototheca moriformis. In some embodiment, triglyceride has been removed from the microalgal cells, and wherein the amount of triglyceride removed from the microalgal cells is more than 10% of the dry weight of the microalgal cells. In some embodiments, the fatty acid composition of the biomass comprises at least 60% C18:1; at least 50% combined total amount of C10, C12, and C14; or at least 70% combined total amount of C16:0 and C18:1. Suitable plasticizers include, e.g., one or more of: glycerol, sorbitol, triacetin, triethyl citrate, acetyl triethyl citrate, tributyl cirtate, acetyl tributyl citrate, trioctyl citrate, acetyl trioctyl citrate, trihexyl citrate, butyryl trihexyl citrate, trimethyl citrate, alkyl sulphonic acid phenyl ester, and 1,2-cyclohexane dicarboxylic acid diisononyl ester. Suitable additional polymers include, e.g., of one or more of: polylactic acid, polycaprolactone, polyesteramide, polyhydroxybutyrate, polyhydroxybutyrate-co-valerate, polyhydroxyalkanoate, polyethylene, polypropylene, polyethylene terephthalate, and polycarbonate. In some embodiments, the biomass is insoluble in an aqueous solvent, said insoluble fraction produced by removing components soluble in an aqueous solvent from oleaginous microbial biomass. In some embodiments, the biomass is a biomass fraction that is insoluble in an aqueous solvent. In some embodiments, the methods further comprise the step of forming the thermoplastic through one or more steps selected from extruding, molding, blowing, coating, and calendering.

[0014] In one embodiment, a thermoset composition of the invention is made by covalently modifying biomass with a phenolic moiety, an isocyanate moiety, an epoxide moiety, or an imide moiety. Phenolized biomass can be prepared by reacting the biomass with a phenol containing reactant in the presence an acidic catalyst, for example, sulfuric acid. The phenolization reaction is typically carried out at temperatures of 50.degree. C. to 200.degree. C. One exemplary phenol containing reactant is benzyl alcohol. Biomass can be covalently modified with isocyanate moieties by reacting the biomass with a compound that contains one or more isocyanate moieties. The reaction is typically carried out at temperatures of 50.degree. C. to 200.degree. C. Exemplary compounds that contain one or more isocyanate moieties include methylene diphenyl diisocyanate (MDI), toluene diisocyanate (TDI), (HDI), isophorone diisocyanate (IPDI), and methyl isocyanate (MIC). The covalently modified isocyanate biomass is then reacted with a polyol to form the thermoset composition. Biomass can be covalently modified to comprise epoxides by reacting the biomass with peroxide containing reactants. The peroxide containing biomass is then subsequently cured to form the thermoset composition. Covalently modified biomass that contains imides can be prepared by reacting the biomass with for example, N,N-dimethylacetamide (DMAc) or N-methylpyrrolidinone (NMP), pyromellitic dianhydride (PMDA), and/or 4-4'oxydianiline.

[0015] In certain embodiments, a further aspect of the invention includes a process for producing triglyceride that entails (a) heterotrophically cultivating microalgal cells in a culture medium including crop-derived sugar so as to produce triglyceride inside the cells; (b) removing the triglyceride from the cells to produce an oil and a residual biomass; (c) hydrothermally carbonizing a water soluble fraction and/or water insoluble fraction of the biomass to produce a carbonized product and a nutrient-rich aqueous solution; and (d) repeating the process with recycling of the nutrients of the nutrient-rich aqueous solution to step (a) to support the cultivation of additional microalgal cells or using the nutrients of the nutrient-rich aqueous solution in the growing of crops. In particular embodiments, the microalgal cells have a mean diameter between approximately 1 micron and approximately 50 microns. In some embodiments, the microalgal cells are obligate heterotrophs. In certain embodiments, removed triglyceride accounts for more than 10% of the dry weight of the microalgal cells. In certain embodiments, the biomass is carbonized in the presence of an acidic catalyst. For example, the biomass can be hydrothermally carbonized by heating it in the presence of water to between about 180-350.degree. C. for between 60 to 180 minutes. In such embodiments, the amount of acidic catalyst can be in the range of 0.01 grams to 0.6 grams per gram of biomass. Suitable acidic catalysts include, e.g., citric acid and acrylic acid. In certain embodiments, the fatty acid composition of the biomass includes at least 60% C18:1; at least 50% combined total amount of C10, C12, and C14; or at least 70% combined total amount of C16:0 and C18:1.

[0016] In certain embodiments, provided is a composition comprising a blend of a moldable polymer, a microalgal biomass, and optionally a lipid selected from a triacylglyceride, a fatty acid, a fatty acid salt, a fatty acid ester, and one or more combinations thereof, wherein the microalgal biomass is optionally covalently modified and is obtained from a heterotrophic oleaginous microalgae. In certain embodiments, provided is a composition comprising a blend of a moldable polymer, a microalgal biomass, and optionally a lipid selected from a triacylglyceride, a fatty acid, a fatty acid salt, a fatty acid ester, and one or more combinations thereof, wherein the microalgal biomass is optionally covalently modified and is obtained from a heterotrophic oleaginous microalgae that is an obligate heterotroph.

[0017] In certain embodiments, provided is a film comprising a composition provided herein.

[0018] In certain embodiments, provided is an injection molded article comprising a composition provided herein.

[0019] In one embodiment, the compositions provided herein do not contain a plant polymer.

[0020] These and other aspects and embodiments are further described in the drawings and detailed descriptions below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The foregoing features of the invention will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawing, in which:

[0022] FIG. 1 shows a flow diagram depicting a method for preprocessing biomass in connection with some embodiments of the present invention.

[0023] FIG. 2 shows scanning electron microscopy (SEM) morphology of selected hydrothermal treated microalgal samples made with an embodiment of the compositions as illustrated in Example 5.

[0024] FIG. 3 shows Fourier transform infrared (FTIR) spectra of selected carbon samples made with an embodiment of the compositions as illustrated in Example 5.

[0025] FIG. 4 shows a graph of charge densities of the crosslinked, anionized biomass made with an embodiment of the compositions as illustrated in Example 7.

[0026] FIG. 5A-B show the retention results of filtration studies conducted on paper preparations made with biomass in an embodiment of the compositions as illustrated in Example 12.

DETAILED DESCRIPTION

Definitions

[0027] "About" refers to the stated value .+-.10%.

[0028] "Acylation" refers to a reaction between a reactant having a hydroxy group and a reactant having activated carbonyl group to produce an ester linkage. Activated carbonyl groups include anhydrides, esters, acids, and acyl groups having a leaving group such as a halide attached to the carbonyl carbon. "Acetylation" refers to an ester producing reaction where one of the reactants has an acetyl (CH.sub.3C.dbd.O--) group.

[0029] "Biomass" is material produced by growth and/or propagation of cells including whole cells, whole cell debris, cell wall material, polysaccharides, triglycerides, proteins, and other intracellular or extracellular components. "Residual biomass" refers to biomass that remains after cells are processed, such as when oil is extracted. In certain embodiments, the biomass comprises 65-50%, 50-30%, 40-20%, 30-10%, 20-10%, and 10-5% of the compositions provided herein.

[0030] "Oleaginous microbial biomass" shall mean biomass derived from oleaginous microbes.

[0031] An "oleaginous" cell is a cell capable of producing at least 20% lipid by dry cell weight, either in its wild-type form or upon recombinant or classical strain improvement. An "oleaginous microbe" or "oleaginous microorganism" is a microbe, including a microalga, that is oleaginous. In some embodiments, the cell produces at least 50%, at least 60%, at least 70%, at least 80%, or at least and 90% triglyceride by dry cell weight.

[0032] The term "bulk properties" in connection with the compositions provided herein refers to any measureable property of the composition, including those properties that are dependent on the size of the composition. Bulk properties include physical, mechanical, thermal, optical, barrier, and related performance properties of the composition. Specific properties include but are not limited to density, impact resistance, tensile strength, flexural strength, seal strength, glass transition temperature, melting point, melt flow index, porosity, thickness, color, brightness, opacity, light scattering, light absorption, roughness, water vapor transition rate, and water absorption. Bulk properties can be tested using conventional methods, such as those published by ASTM (American Society for Testing and Materials) International, TAPPI Standards, Scandinavian Pulp, Paper and Board Testing Committee (SCAN-C) and International Organization for Standardization (ISO). In some embodiments, the bulk properties of the composition differ in comparison to the bulk properties of the moldable polymer alone by 25% or less. In some embodiments, one of the bulk properties is increased by 10% or less. In other embodiments, one of the bulk properties is decreased by 10% or less.

[0033] The term "moldable polymer" refers to moldable synthetic or semi-synthetic polymers for use in plastics. The moldable polymers may be amorphous or semicrystalline, and include thermoplastic and thermosetting polymers. In some embodiments, the moldable polymer is also a biodegradable polymer.

[0034] In connection with a biomass derived material, "thermoplastic" shall mean a material or composition that is thermoplastic or is thermoplastic-like in that, in the presence of a plasticizer, elevated temperatures, and/or shearing, it melts and fluidizes, enabling its use in preparing articles traditionally made with thermoplastics. In one embodiment, microbial biomass is subjected to elevated temperatures and shearing in the presence of a plasticizer (e.g. a known thermoplastic) to form thermoplastics or blends thereof. In the softened state, the thermoplastic material can be formed into a finished product. Often, the thermoplastic material is first made into pellets, blocks or other convenient size; the pellets or blocks are re-softened, typically by heating, and shaped into a finished product.

[0035] "Thermoset" shall mean a material or composition that cures or hardens into a desired shape by the application of heat, raditaion (e.g., ultraviolet light, laser radiation, etc.) or other energy sources to the material, or by a chemical reaction. Prior to curing, thermoset materials are malleable and can be molded into a desired form. Once cured, the thermoset material cannot be softened and remolded to a different form. The curing process transforms the material by a cross-linking process.

[0036] "Colored molecules" or "color generating impurities" as used herein refer to any compound that imparts a color to the extracted oil. "Colored molecules" or "color generating impurities" include for example, chlorophyll a, chlorophyll b, lycopenes, tocopherols, campesterols, tocotrienols, and carotenoids, such as beta carotene, luteins, zeaxanthin, astaxanthin. These molecules are preferably present in the microbial biomass or the extracted oil at a concentration of no more than 500 ppm, no more than 250 ppm, no more than 100 ppm, no more than 75 ppm, or no more than 25 ppm. In other embodiments, the amount of chlorophyll that is present in the microbial biomass or the extracted oil is less than 500 mg/kg, less than 100 mg/kg, less than 10 mg/kg, less than 1 mgkg, less than 0.5 mg/kg, less than 0.1 mg/kg, less than 0.05 mg/kg, or less than 0.01 mg/kg.

[0037] "Cultivated", and variants thereof such as "cultured" and "fermented", refer to the intentional fostering of growth (increases in cell size, cellular contents, and/or cellular activity) and/or propagation (increases in cell numbers) of one or more cells by use of selected and/or controlled conditions. The combination of both growth and propagation is termed "proliferation." Examples of selected and/or controlled conditions include the use of a defined medium (with known characteristics such as pH, ionic strength, and carbon source), specified temperature, oxygen tension, carbon dioxide levels, and growth in a bioreactor. "Cultivated" does not refer to the growth or propagation of microorganisms in nature or otherwise without human intervention; for example, natural growth of an organism that ultimately becomes fossilized to produce geological crude oil is not cultivation. In some embodiments, microbes such as microalgae are cultivated on sugar from corn, sorghum, sugar cane, sugar beet, or molasses. In other embodiments the microbes are cultivated on sucrose.

[0038] "Covalently modified" shall mean microbial biomass wherein the polysaccharides, the proteins, or the triacylglycerols within the microbial biomass have been covalently modified with a hydrophobic group, a hydrophilic group, an anionic group or a cationic group prior to the formation of the thermoplastic material. During the thermoplastic forming process, components of the microbial biomass, for example, polysaccharides, proteins, and/or triacylglycerols, may be further covalently modified by exposure of the microbial biomass to heat, shearing and plasticizer.

[0039] "Lipid" refers to fatty acids and their derivatives, including free fatty acids and their salts, as well as fatty acid esters. Fatty acid esters include fatty acid alkyl esters and triacylglycerides. Fatty acid salts include sodium, potassium, magnesium, and calcium salts. Fatty acids can be referred to by shorthand notation "carbon number:number of double bonds". Thus C18:1 refers to an 18 carbon fatty acid chain having one double bond. In certain embodiments, the lipids provided herein comprise 15%, 10%, 5%, or 2% or less of the plastic and film compositions provided herein. In other embodiments the lipid is a calcium salt. In still other embodiments the lipid has at least 60% C18:1; or at least 50% combined total amount of C10, C12, and C14; or at least 70% combined total amount of C16:0 and C18:1.

[0040] "Fatty acid profile" refers to the distribution of fatty acids in a cell or oil derived from a cell in terms of chain length and/or saturation pattern. In this context the saturation pattern can comprise a measure of saturated versus unsaturated acid or a more detailed analysis of the distribution of the positions of double bonds in the various fatty acids of a cell. Unless specified otherwise, the fatty acid profile is expressed as a weight percent of the total fatty acid content.

[0041] "Lysis" is the breakage of the plasma membrane and optionally the cell wall of a biological organism sufficient to release at least some intracellular content, often by mechanical, chemical, viral or osmotic mechanisms that compromise its integrity. "Lysing" is the process of lysis.

[0042] "Microalgae" is a microbial organism that contains a chloroplast or plastid, and optionally that is capable of performing photosynthesis, or a prokaryotic microbial organism capable of performing photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed carbon source as energy, as well as heterotrophs, which can live solely off of a fixed carbon source. Microalgae include unicellular organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, as well as microbes such as, for example, Volvox, which is a simple multicellular photosynthetic microbe of two distinct cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae also include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae also include obligate heterotrophic microorganisms that have lost the ability to perform photosynthesis, such as certain dinoflagellate algae species and species of the genus Prototheca. In some embodiments the microalgae is a Parachlorella, Prototheca, Chlorella or strains having at least 85% nucleotide sequence identity in 23S rRNA sequences to a Parachlorella, Prototheca, or Chlorella strain. Certain nucleic acid sequences are disclosed in WO2009/126843 which is incorporated herein by reference in its entirety. Such sequences in WO2009/126843 include SEQ ID NOs:3-29.

[0043] The term "sugar" in connection with algal feedstock refers to carbohydrates that are derived from natural sources or that are synthetically or semi-synthetically prepared. Sugar can be derived from natural sources such as through extraction (e.g. sugarcane or sugar beet) or by further chemical, enzymatic processing (e.g. sugar from corn), and/or by depolymerizaton of cellulosic materials.

[0044] The present invention is based on the realization that biomass, particularly residual biomass that remains after cell lysis, especially of microalgae cultured heterotrophically, is a valuable product, the utilization of which confers substantial overall economic advantage to using the cells as production organisms for making fatty acids or other high value products. Indeed, the economic advantage gained may outweigh the expense associated with the lysis of the cell walls. Judicious use of the residual biomass may compensate for loss of efficiency in the process resulting from conversion of sugar and cell-energy to cell wall synthesis rather than toward production of the desired product. Embodiments of the invention also allow for recovery and potential recycling of valuable nutrients used in the culture of the microalgae, including phosphorous, potassium, and nitrogen. The materials so formed may have the added advantage of being biodegradable.

[0045] Furthermore, by using single-celled oleaginous microbial biomass, such as microalgal biomass, particles, comprising polysaccharides and/or proteins, having a size distribution that is believed to be unobtainable or difficult to obtain from multicellular sources of biomass (e.g., higher plants or multicellular algae) is obtained. For example, cells of oil-bearing Prototheca moriformis may have a tight size distribution around about 10 micron diameter. Cells of the microalgal biomass typically have a mean diameter between approximately 1 micron and approximately 50 microns. In certain cases the mean diameter ranges between approximately 2 microns and 40 microns, 3 microns and 30 microns, 4 microns and 20 microns or 5 microns and 15 microns.

[0046] After lysis and extraction of the oil, the residual biomass including the cell wall material may have a similarly tight size distribution. The size of the particles obtained, their distribution, the amount of residual oil remaining after oil extraction, and/or the protein or saccharide composition of biomass may confer previously unknown advantages to the products or process described herein. By contrast, the processing of fibers produced by higher plants may not afford the same particle size distribution. In one embodiment, the oleaginous microbial biomass, prior to lysis and extraction of the triacylglycerides, have a similar tight size distribution.

[0047] In one embodiment, the specific gravity of a thermoplastic or thermoset composition does not increase or does not significantly increase upon blending a polymer with single-celled oleaginous microbial biomass, such as microalgal biomass. Low or no increases in specific gravity is a desirable benefit when blending polymers with biomass for specific applications requiring light weight components. In some embodiments, the specific gravity of a thermoplastic or thermoset composition increases by less than 10%, less than 5%, less than 2%, or less than 1% when as much as 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% by weight of a thermoplastic polymer is replaced with single-celled oleaginous microbial biomass, such as microalgal biomass, to form a thermoplastic or thermoset blend.

[0048] In particular, the following methods for treating biomass to increase its value are disclosed below: (i) acetylation of microalgal biomass to produce a material useful in the production of thermoplastics; (ii) use of triglyceride containing microalgal biomass for production of thermoplastics; (iii) combination of microalgal biomass and at least one type of plant polymer to produce a material useful in the production of thermoplastics; (iv) anionization of microalgal biomass to form a water absorbant material; (v) cationization of microalgal biomass, and optional flocculation, to form a water absorbant material; (vi) crosslinking of anionized microalgal biomass; (vii) carbonization of microalgal biomass; and (viii) use of microalgal biomass in the making of paper.

[0049] In addition, products produced by these processes and uses thereof are disclosed.

[0050] Production of Biomass.

[0051] For all of the embodiments presented herein, the cells may be grown heterotrophically as disclosed in (step 100). Although the cells may be individual plant cells (i.e., cells grown in culture), microbial cells are preferred. Microalgae may be grown heterotrophically as described in WO2008/151149 and WO2010/063032. The microalgae can also be an obligate heterotroph.

[0052] In various embodiments of the invention, the biomass is prepared by fermentation of a microbe selected from the group consisting of microalgae, oleaginous bacteria, oleaginous yeast, and fungi. In various embodiments, the microalgae is a species of a genus selected from Chlorella, Parachlorella, or Prototheca, or is one of the other species in Table 1. In various embodiments, the oleaginous bacteria is a species of the genus Rhodococcus. In various embodiments, the oleaginous yeast is Rhodosporidium toruloides or another species listed in Table 2. In various embodiments, the fungus is a species listed in Table 3.

[0053] In various embodiments, the microalgae are of the genera Chlorella and Prototheca, including Chlorella protothecoides and Prototheca moriformis, which are capable of accumulating substantial amounts of triglyceride (e.g., 50 to 85% by dry cell weight). In an embodiment of the present invention, the microorganism is of the genus Chlorella, preferably, Chlorella protothecoides, Chlorella ellipsoidea, Chlorella minutissima, or Chlorella emersonii. Chlorella is a genus of single-celled green algae, belonging to the phylum Chlorophyta. It is spherical in shape, about 2 to 10 .mu.m in diameter, and is without flagella. Some species of Chlorella are naturally heterotrophic. In an embodiment of the present invention, the microorganism is of the genus Prototheca, which are obligate heterotrophs.

TABLE-US-00001 TABLE 1 Microalgae. Achnanthes orientalis, Agmenellum, Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis linea, Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphora coffeiformis tenuis, Amphora delicatissima, Amphora delicatissima capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteoccocus aerius, Bracteococcus sp., Bracteacoccus grandis, Bracteacoccus cinnabarinas, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora (strain SAG 37.88), Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella cf. minutissima, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides (including any of UTEX strains 1806, 411, 264, 256, 255, 250, 249, 31, 29, 25), 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 f. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena, Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium (UTEX LB 2614), Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Neochloris oleabundans, Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, 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 beijerinckii, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamii, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Sarcinoid chrysophyte, Scenedesmus armatus, Scenedesmus rubescens, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

TABLE-US-00002 TABLE 2 Oleaginous Yeast. Candida apicola, Candida sp., Cryptococcus curvatus, Cryptococcus terricolus, Debaromyces hansenii, Endomycopsis vernalis, Geotrichum carabidarum, Geotrichum cucujoidarum, Geotrichum histeridarum, Geotrichum silvicola, Geotrichum vulgare, Hyphopichia burtonii, Lipomyces lipofer, Lypomyces orentalis, Lipomyces starkeyi, Lipomyces tetrasporous, Pichia mexicana, Rodosporidium sphaerocarpum, Rhodosporidium toruloides Rhodotorula aurantiaca, Rhodotorula dairenensis, Rhodotorula diffluens, Rhodotorula glutinus, Rhodotorula glutinis var. glutinis, Rhodotorula gracilis, Rhodotorula graminis Rhodotorula minuta, Rhodotorula mucilaginosa, Rhodotorula mucilaginosa var. mucilaginosa, Rhodotorula terpenoidalis, Rhodotorula toruloides, Sporobolomyces alborubescens, Starmerella bombicola, Torulaspora delbruekii, Torulaspora pretoriensis, Trichosporon behrend, Trichosporon brassicae, Trichosporon domesticum, Trichosporon laibachii, Trichosporon loubieri, Trichosporon loubieri var. loubieri, Trichosporon montevideense, Trichosporon pullulans, Trichosporon sp., Wickerhamomyces Canadensis, Yarrowia lipolytica, and Zygoascus meyerae.

TABLE-US-00003 TABLE 3 Oleaginous Fungi. Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, Hensenulo, Chaetomium, Cladosporium, Malbranchea, Rhizopus, and Pythium.

[0054] The microalgae may be genetically engineered by introducing an exogenous gene so as to allow the cells utilize an alternate sugar and/or to alter the chain length and saturation profiles of the fatty acids produced by the microalgal cells. For example the cells may use sucrose (e.g., from sugar cane, beets or palm) by recombinant introduction of an exogenous secreted sucrose invertase gene, chain length distribution may be altered through the introduction of an exogenous acyl-ACP thioesterase and/or reduction of endogenous acyl-ACP thioesterase activity (e.g., knockout or knockdown), and saturation profile may be altered through the introduction of an exogenous fatty acid desaturase and/or reduction of endogenous desaturase activity (e.g., knockout or knockdown).

[0055] In some embodiments, color-generating compounds (e.g., carotenoids) are present in the microbial biomass at a concentration of no more than 6000 ppm, no more than 5000 ppm, no more than 4000 ppm, no more than 3000 ppm, no more than 2000 ppm, no more than 1000 ppm, 500 ppm, no more than 250 ppm, no more than 100 ppm, no more than 75 ppm, or no more than 25 ppm. Color-generating compounds include carotenoids such as lutein, beta carotene, zeaxanthin, astaxanthin and chlorophyll. In other embodiments, the amount of chlorophyll that is present in the microbial biomass is less than 3500 ppm, less than 3000 ppm, less than 2500 ppm, less than 2000 ppm, less than 1500 ppm, less than 1000 ppm, less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 25 ppm, less than 10 ppm, less than 5 ppm, less than 1 ppm. The amount of chlorophyll that is present in the microbial biomass can range from, e.g., 0.1 ppm to 3000 ppm; this range can be bounded by any of the values in the previous sentence.

[0056] Optionally, by using biomass produced from heterotrophically cultivated cells, the resulting compositions may have less color, especially green color, due to lack of chlorophyll. As a result, reduced bleaching or use of lesser amounts of colorants may be required to achieve an article with an acceptable color. Color characteristics may be analyzed by quantification of color according to methods utilizing a three-component theory of color vision. In colorimetry, these components are referred to as X-Y-Z coordinates. Alternatively or in addition, color characteristics may be quantified through the use of spectrophotometry or other methods known in the art.

[0057] When processed into compositions such as thermoplastics, thermosets, absorbents, adsorbents, or paper, algal biomass derived from microalgae or microalgae cultivated photosynthetically, such as in ponds, swamps, waste water treatment facilities, or photobioreactors impart a visually unappealing green color to the composition and/or have an unpleasant fishy or seaweed odor. In specific embodiments, the oleaginous microorganism can be cultivated heterotrophically, in the dark. The cells of the microorganism can have less than 2.5% DHA (docosahexaenoic acid); less than 3000 ppm chlorophyll; less than 5000 ppm of color generating compounds; and/or be lacking in an unpleasant odor.

Extraction of Triglycerides.

[0058] After growing the cells, triglycerides may be extracted (step 110). Methods for oil extraction, pressing, and cell lysis are given in WO2008/151149, WO2010/063032, WO2010/120939, and WO2010/138620. Oil may be extracted (step 120) by one or more of mechanical pressing, solvent (e.g., hexane) extraction, sonication, or other suitable method. Mechanical pressing methods may optionally include addition of press aid. For example, WO2010/120939 teaches a device and method for pressing of oil from microalgae using a press-aid (also referred to therein as a "bulking

» Number: 20130236937

» Publication Date: 12/09/2013

» Applicant: > Assignee Name and Adress:

» Inventor: Harlin; Ali; (Espoo, FI) ; Jaaskelainen; Anna-stiina; (Espoo, FI) ; Kiuru; Jani; (Espoo, FI) ; Laine; Christiane; (Espoo, FI) ; Liitia; Tiina; (Espoo, FI) ; Nattinen; Kalle; (Espoo, FI) ; Pere; Jaakko; (Espoo, FI) ; Sousa; Sonia; (South San Francisco, C

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