WO2015196134A1 - Wood composites - Google Patents

Abstract

Provided are engineered wood materials containing oleaginous microbial biomass, methods for their preparation, and uses thereof. These materials include wood composites and wood plastic composites.

Description

WOOD COMPOSITES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S. C. 119(e) of US Provisional Patent Application No. 62/015,154, filed June 20, 2014, US Provisional Patent Application No. 62/064,381, filed October 15, 2014, US Provisional Patent

Application No. 62/073,728, filed October 31, 2014, and US Provisional Patent Application No. 62/094,734, filed December 19, 2014, each of which is incorporated herein by reference in its entirety.

INTRODUCTION Engineered wood such as wood composites and wood plastic composites are environmentally beneficial alternatives to solid wood. These composites minimize reliance on virgin timber sourced from slow growing forests. The composites also allow mill waste and low value wood by-products generated during lumber production to be transformed into useful substitutes for solid wood. Examples of manufactured wood composites that are found in many everyday products include particle boards, medium density fiber boards, and oriented strand boards. These materials are increasingly in demand, as are wood plastic composites for use as low maintenance alternatives to solid wood.

SUMMARY In an embodiment, the present invention provides engineered wood materials comprising oleaginous microbial biomass. In some embodiments, the biomass is optionally thermochemically treated.

In one embodiment, provided is a wood plastic composite comprising a) a blend of a thermoplastic resin; b) a cellulosic filler; c) an oleaginous microbial biomass, and d) a binder or a coupling agent, wherein the biomass is optionally thermochemically treated. In some embodiments, the biomass is thermochemically treated.

In some embodiments, provided is a wood plastic composite comprising a blend of

a) 30 to 70% by weight of a thermoplastic resin; b) 30 to 70% by weight of a cellulosic filler and an oleaginous microbial biomass, and c) 0.1 to 5%> by weight of a coupling agent.

In some embodiments, provided is a wood plastic composite comprising a blend of

a) 40 to 60%) by weight of a thermoplastic resin;

b) 30 to 60%o by weight of a cellulosic filler and an oleaginous microbial biomass, and c) 0.1 to 5%) by weight of a coupling agent.

In some embodiments, provided is a wood plastic composite comprising a blend of

a) 30 to 45%) by weight of a thermoplastic resin;

b) 50 to 55%o by weight of a cellulosic filler and an oleaginous microbial biomass, and c) 0.1 to 5%o by weight of a coupling agent.

In some embodiments, the oleaginous microbial biomass is present in an amount up of to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%> by weight of the composite. In some embodiments, the oleaginous microbial biomass is present in an amount up of to 5 to 10% by weight of the composite. In other embodiments, the oleaginous microbial biomass is present in an amount up of to 5% by weight of the composite. In still other embodiments, the oleaginous microbial biomass is present in an amount up of to 10% by weight of the composite. In another embodiment, provided is a method for preparing a wood plastic composite, the method comprising a) blending a thermoplastic or thermoset resin, a cellulosic filler, an optionally thermochemically treated oleaginous microbial biomass, and a binder or a coupling agent to form a mixture; and b) extruding, injection molding, hot-pressing, or calendaring said mixture to form the wood plastic composite. In some embodiments, the biomass is thermochemically treated.

In some embodiments, the thermoplastic resin is selected from the group consisting of a polystyrene, polyolefm, polyvinyl chloride, polylactic acid, and polymethyl methacrylate resin. In other embodiments, the polyolefm is recycled. In still other embodiments, the polyolefm is polyethylene or polypropylene. In some embodiments, the polyethylene is low density polyethylene (LDPE), high density polyethylene (HDPE), or recycled HDPE. In other embodiments, the thermoplastic resin comprises up to 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85% by weight of the composite.

In some embodiments, the coupling agent is a silane, a maleic anhydride grafted polyolefm, succinic acid, or a succinic anhydride. In other embodiments, the coupling agent is maleic anhydride grafted high density polyethylene (MAPE) or maleic anhydride grafted polypropylene (MAPP). In other embodiments the coupling agent is an organic acid, a di-acid, a tri-acid, an anhydride, a cyclic anhydride, or boric acid. In still other embodiments, the coupling agent is glutaric acid, glycolic acid, oxalic acid, citric acid, or adipic acid. In one embodiment, provided is a wood composite comprising a blend of: a) a cellulosic filler and

b) an oleaginous microbial biomass,

c) a binder;

wherein the wood composite is not a wood plastic composite. In some embodiments, the biomass is thermochemically treated.

In another embodiment, provided is a method for preparing a wood composite, the method comprising a) blending a cellulosic filler, an oleaginous microbial biomass, and a binder to form a mixture; and b) extruding, injection molding, hot- pressing, or calendaring said mixture to form the wood composite, wherein the wood composite is not a wood plastic composite.

In some embodiments, the binder is urea formaldehyde, phenol formaldehyde, melamine, an isocyanate, polymeric diphenyl methane diisocyanate (polymeric MDI), an emulsion polymer isocyanate, resorcinol, phenol resorcinol, or an epoxy resin. In other embodiments, the binder is an isocyanate, methylene diphenyl diisocyanate (MDI), or polymeric methylene diphenyl diisocyanate. In still other embodiments, the binder is polyvinyl acetate, a polyurethane, a polyurethane/emulsion polymer, an elastomer, a hot-melt, starch, casein, blood, or animal glue.

The wood composites and wood plastic composites can also have one or more of the following features. In some embodiments, the binder or coupling agent is present in an amount of up to 0.25, 0.5, 1, 2, 3, 4, or 5% by weight of the composite. In some embodiments, the binder in the wood composite is reduced by at least 90%, 80%), 70%), 60%), or 50%> compared to a wood composite not containing an oleaginous microbial biomass. In other embodiments, the binder in the wood composite is reduced by at least 45%, 40%>, 35%, 30%>, 25%, or 20%> compared to a wood composite not containing an oleaginous microbial biomass.

In some embodiments, the oleaginous microbial biomass is delipidated. The oleaginous microbial biomass can be delipidated such as by pressing or solvent extraction. In other embodiments, the biomass is extracted with hexanes. In still other embodiments, the biomass is extracted with ethanol or mixtures of ethanol and water.

In some embodiments, the delipidated biomass comprises residual lipid that is present in an amount of up to 20%>, 19%>, 18%, 17%, or 16% by weight of the biomass. In some embodiments, the delipidated biomass comprises residual lipid that is present in an amount of up to 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%), 4%), 3%), 2%> or 1% by weight of the biomass. In other embodiments, the residual lipid is present in these amounts following thermochemical treatment such as by torrefaction. In some embodiments, the residual lipid comprises predominantly triglycerides. In some embodiments, provided is a wood composite comprising residual lipids. In other embodiments the residual lipids facilitate the manufacture of a wood composite such as a by increasing the speed at which the composite can be formed on the production line.

In some embodiments, the delipidated biomass optionally comprises a press aid. In other embodiments, the press aid is soy hulls. In certain embodiments the press aid is pyrolyzed. In some embodiments the press aid is torrefied. In some embodiments, the press aid is present in an amount of up to 55%,

50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% by weight of the delipidated biomass.

In some embodiments, the biomass optionally containing press aid is present in an amount of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20% by weight of the composite. In some embodiments, the biomass optionally containing press aid is ground. In certain embodiment, the biomass is ground to less than 3500 microns. In other embodiments, the biomass optionally containing press aid is milled. In still other embodiments, the biomass optionally containing press aid has an average particle size of from 0.1 to 1000 microns. In certain embodiments, the biomass has an average particle size about 500 to 350 microns. In other embodiments, the biomass has an average particle size about 500 to 250 microns. The some embodiments the biomass contains press aids prior to grinding or milling.

In some embodiments, the biomass optionally containing press aid is pyrolyzed. In certain embodiments, the biomass is subjected to fast pyrolysis.

In some embodiments, the biomass optionally containing press aid is torrefied.

In some embodiments, the biomass comprises an oleaginous bacteria, oleaginous yeast, or oleaginous microalgae.

In some embodiments, the biomass comprises a heterotrophic oleaginous microalgae.

In some embodiments, the microalgae is cultivated with sugar from corn, sorghum, sugar cane, sugar beet, or molasses as a carbon source.

In some embodiments, the microalgae is cultivated on sucrose.

In some embodiments, the microalgae is Parachlorella, Prototheca,

Auxenochlorella protothecoides, or Chlorella. In certain embodiments, the microalgae is Prototheca moriformis .

In some embodiments, the composite provided herein comprises oil produced by the microalgae.

In some embodiments, the microalgae produces an oil having a fatty acid profile of at least 60% C18: l; or at least 50% combined total amount of CIO, C12, and C14; or at least 70% combined total amount of C16:0 and C18: l .

In some embodiments, the oil produced by the microalgae has a fatty acid profile of at least 80-85% CI 8: 1.

In some embodiments, the oil produced by the microalgae has a fatty acid profile of less than 1 % or 0.1 % polyunsaturated fatty acids. In some embodiments, the cellulosic filler is selected from the group consisting of a wood fiber, a wood flour, paper, coconut flour, coffee flour, rice hull, bamboo, and soy hull or combinations thereof. In other embodiments, the cellulosic filler is a wood fiber, flour, or chip. In still other embodiments, the cellulosic filler is eucalyptus or is an oak, pine, or maple wood fiber, flour, or chip.

In some embodiments, the cellulosic filler is present in an amount of up to 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65% of the composite.

In some embodiments, the composite provided herein further comprises a lubricant. The lubricants assist in the processing of the composite, such as during extrusion. The lubricants allow for faster processing times and/or improve the appearance and uniformity of the wood product. In other embodiments, the lubricant is a metallic or non-metallic stearate. In certain embodiments, the lubricant is a zinc stearate. In some embodiments, the lubricant is a polyester wax, a paraffin wax, a polypropylene wax, a fatty acid derived diamide, ethylene bis-oleamide, a stearate ester, mixed fatty acid esters or amides, or combinations thereof. Suitable lubricants include commercially available lubricants such as Struktol TPW104. In other embodiments, the lubricant is ethylene bis-stearamide.

In some embodiments, the composite provided herein further comprises one or more of a mineral filler, a plasticizer, a UV stabilizer, a colorant, a pesticide, a density modifier, an-antioxidizing agent, and a pesticide. In certain embodiments, the pesticide is one or more of an anti-microbial agent, a fungicide, and an insecticide. In other embodiments, the mineral filler is talc or mica or a combination thereof. In certain embodiments, the mineral filler is talc or a combination thereof.

In some embodiments, provided is an article comprising a composite disclosed herein. In some embodiments, the article is selected from the group consisting of flooring material, outdoor decking, wood paneling, window framing material, interior trim material, railing, fencing, siding, shingles, roofing materials, and an automotive part.

In some embodiments, composite is encased in a capstock. The capstock forms an outer layer on at least a portion of the composite. Particularly in an outdoor setting, the capstock acts to protect the inner core from exposure to the environment such as to moisture, mold, and mildew. In other embodiments the capstock comprises a thickness of about 0.012 inches to about 0.040 inches. In certain embodiments the capstock comprises a thickness of about 0.015 inches to about 0.020 inches. The capstock can be formed as disclosed in US 2010/0159213 such as by co-extrusion. In some embodiments the capstock comprises an ionomer. Suitable ionomers are selected from ethylene/methacrylic acid copolymers and zinc ionomers thereof. The capstock can also be formed as disclosed in US 2012/0315471, wherein the capstock comprises at least one of an elastomer and a plastomer. In some embodiments the elastomer comprises at least one of a propylene based elastomer, an ethylene propylene diene monomer, a three block thermoplastic elastomer, and a two block thermoplastic elastomer. In other embodiments the plastomer comprises at least one of a metallocene, very low density polyethylene, polyethylene, and ethylene methacrylate. In still other embodiments the capstock further comprises one or more of a colorant, a variegated colorant, a UV stabilizer, an antioxidant, an antistatic agent, a biocide, and a fire retardant.

These and other embodiments and features of the invention are further described in the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows extruded profiles of wood plastic composite compositions in connection with biomass in an embodiment of the compositions as illustrated in Example 3.

Fig. 2 shows extruded profiles of wood plastic composite compositions in connection with biomass in an embodiment of the compositions as illustrated in Example 4.

DETAILED DESCRIPTION

DEFINITIONS

"About" refers to the stated value + 10%.

"Binder" refers to adhesives for binding cellulosic fillers. Binders are used in wood composite and, although less common, can also be used in wood plastic composites in place of coupling agents. In some embodiments the binder is a synthetic resin such as a thermosetting binder. In some embodiments, the composite includes one or more thermosetting binders such as urea formaldehyde, phenol formaldehyde, melamine, polymeric diphenyl methane diisocyanate (polymeric MDI), emulsion polymer isocyanate, resorcinol, phenol resorcinol, and epoxy resin. In some embodiments, the binder is a thermoplasic binder such as polyvinyl acetate, polyurethanes, polyurethane/emulsion polymer, elastomers, and hot-melts. In other embodiments, the binder is a natural adhesive such as starch, casein, blood, and animal glues.

"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. The biomass can contain any left-over components of the fermentation broth including sugars and minerals. The biomass can also contain press aids if these are used to extract the oil, as well as any residual oil not removed during pressing. In some embodiments the biomass comprises 5-55% by weight press aid. In certain embodiments, the biomass comprises 65-50 %, 50-30 %, 40-20 %, 30-10 %, 20-10 %, and 10-50 % of the compositions provided herein.

"Cellulosic filler" as used herein refers to cellulose containing fillers.

Cellulosic fillers are typically derived from lignocellulosic material which contain cellulose, hemicellulose, and lignin as the principle structural components. Cellulosic fillers can be fibrous having a greater length than cross section (e.g. a fiber) or a particle having a generally uniform dimensions (e.g. a flour). In some embodiments, cellulosic filler refers to a mixture of different types of cellulosic filler or shapes (e.g. fiber and flour mixture). Exemplary cellulosic fillers include wood such as from hardwoods and softwoods. Wood types include poplar, maple, oak, pine, birch, spruce, and bamboo. Other wood types include eucalyptus. The wood can be in various forms such as wood chips, bark, sawdust, wood flour, and wood fiber. Wood can be obtained from virgin sources (e.g. forest, tree plantations), recycled sources (e.g. newspapers, recycled lumber) or cutting waste such as from milling or from the timber industry. Other cellulosic fillers include grasses, rice, wheat, and barley straw, sugarcane bagasse, corn stover, corn fiber, and switchgrass. The cellulosic fillers can be pre -treated chemically and/or physically before blending with the microalgal biomass provided herein. The treatment can remove moisture and/or components of the cellulosic fillers such as sugars and polysaccharides. The fillers can also be pulverized or ground such as by hammer milling and screened for specific sizes. In some embodiments, the cellulosic filler is a wood fiber or flour having a size of 80 to 40 mesh. In other embodiments, the wood fiber or flour is 40 to 20 mesh. In still other embodiments, the wood fiber or flour is 40 mesh.

"Coupling agent" refers to agents that reduce the interfacial tension between the hydrophilic cellulosic filler and the hydrophobic thermoplastic in wood plastic composites thereby allowing greater dispersion and stronger bonding between the two components. The term coupling agent as used herein includes compatibilizers and dispersing agents. Coupling agents can provide covalent bonding and/or hydrogen bonding to the hydrophilic phase and polymer chain entanglement with the hydrophobic phase. Agents that covalently bond to the hydrophilic phase include maleated polypropylene (MAPP), maleated styrene-ethylene/butylene-styrene (SEBS- MA), styrene-maleic anhydride (SMA), succinic acid, and organic silanes.

Compatibilizers include acetic anhydride and methyl isocyanate that reduce the hydrophilicity of the cellulosic filler. Dispersing agents create new interfaces between the hydrophilic and hydrophobic phases and prevent or minimize aggregation of the two phases. Suitable dispersing agents include stearic acid and their salts.

"Oleaginous microbial biomass" refers to biomass derived from oleaginous microbes.

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 microalgae, 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.

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, odor, brightness, opacity, light scattering, light absorption, roughness, bumpiness, 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.

"Wood composites" as used herein refers to composites containing cellulosic fillers that are adhered together with a binder. Wood composites differ from wood plastic composites in that the cellulosic fillers are not blended with a thermoplastic where the thermoplastic serves as a partial wood replacement and not as an adhesive. "Thermoset wood composites" refer to wood composites wherein the binder is a thermosetting resin that cures when exposed to the appropriate heat and pressure. Examples of wood composites include glued laminated timber (glulam), oriented strand board (OSB), plywood, particle board, wood fiber insulation board, medium density fiber board (MDF), I-joists, end-jointed lumber, and structural composite lumber (SCL) such as laminated strand lumber (LSL), parallel strand lumber (PSL), laminated veneer lumber (LVL), and oriented strand lumber (OSL). In some embodiments, the wood composites find use in construction such as for furniture, cabinets, countertops, and tiling, paneling, and molding in buildings. In some embodiments the cellulosic filler in the wood composite is eucalyptus.

"Wood plastic composites" as used herein refers to composites containing a blend of cellulosic fillers and thermoplastic resins where the thermoplastic resins serves to replace a portion of the cellulosic filler. Partial replacement can have cost advantages as cellulosic filler can be expensive and difficult to obtain relative to the thermoplastic resin. Additionally, the inclusion of thermoplastic resins can be advantageous in increasing weatherability and durability (e.g. shrinking, warping, splintering, discoloration), particularly in applications where there is exposure to water and sunlight.

In connection with a wood plastic composite, "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.

"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, tocopherols, sterols, tocotrienols, phycobilins, xanthophylls, and carotenoids such as beta carotene, luteins, zeaxanthin, astaxanthin, and lycopene. 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 mg.kg, 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.

"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. "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

numbennumber of double bonds". Thus CI 8: 1 refers to an 18 carbon fatty acid chain having one double bond. In certain embodiments, the lipids provided herein comprise 15%, 10%), 5%o, 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: l; or at least 50% combined total amount of CIO, C12, and C14; or at least 70% combined total amount of CI 6:0 and CI 8: 1. A "fatty acid profile" is the distribution of fatty acyl groups in the triglycerides of the oil without reference to attachment to a glycerol backbone. Fatty acid profiles are typically determined by conversion to a fatty acid methyl ester (FAME), followed by gas chromatography (GC) analysis with flame ionization detection (FID). The fatty acid profile can be expressed as one or more percent of a fatty acid in the total fatty acid signal determined from the area under the curve for that fatty acid. FAME- GC-FID measurement approximate weight percentages of the fatty acids. A "sn-2 profile" is the distribution of fatty acids found at the sn-2 position of the

triacylglycerides in the oil. A "regiospecific profile" is the distribution of

triglycerides with reference to the positioning of acyl group attachment to the glycerol backbone without reference to stereospecificity. In other words, a regiospecific profile describes acyl group attachment at sn-1/3 vs. sn-2. Thus, in a regiospecific profile, POS (palmitate-oleate-stearate) and SOP (stearate-oleate-palmitate) are treated identically. A "stereospecific profile" describes the attachment of acyl groups at sn-1, sn-2 and sn-3. Unless otherwise indicated, triglycerides such as SOP and POS are to be considered equivalent. A "TAG profile" is the distribution of fatty acids found in the triglycerides with reference to connection to the glycerol backbone, but without reference to the regiospecific nature of the connections. Thus, in a TAG profile, the percent of SSO in the oil is the sum of SSO and SOS, while in a regiospecific profile, the percent of SSO is calculated without inclusion of SOS species in the oil. In contrast to the weight percentages of the FAME-GC-FID analysis, triglyceride percentages are typically given as mole percentages; that is the percent of a given TAG molecule in a TAG mixture. Unless specified otherwise, the fatty acid profile is expressed as a weight percent of the total fatty acid content.

"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, enzymatic, viral, or osmotic mechanisms that compromise its integrity. "Lysing" is the process of lysis.

"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 rR A 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.

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 depolymerization or pre-treatment of cellulosic materials.

The term "thermochemically treated", "thermochemical conversion", and "thermochemical decomposition" refers to exposure of biomass to elevated temperatures in the absence of oxygen or under substantially oxygen-free conditions, such as in an atmosphere containing less than 6% or 3% oxygen by volume. In certain embodiments, the thermochemical conversion of biomass disclosed herein occurs at temperatures ranging from 350-500° C. In other embodiments, the thermochemical conversion of the biomass occurs at temperatures ranging from 450- 600° C. Thermochemical conversion produces a solid char and removes moisture and volatile gases from the biomass.

"Pyrolysis" refers to a form of thermochemical conversion at temperatures typically ranging from about 200-1400°C. "Fast pyrolysis" refers to an accelerated form of pyrolysis, where temperatures typically ranging from 350-600° C for 0.5-2 seconds are typically employed. In some embodiments, the biomass is subjected to pyrolysis for a period of less than 10 seconds. In other embodiments, pyrolysis occurs from 0.1 to 5 hours.

"Torrefaction" refers to a form of pyrolysis at temperatures ranging typically from 200 to 320°C. The process can be conducted for a period of 0.1 hours to about 10 hours. Production of biomass.

In various embodiments, 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 I. 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 II. In various embodiments, the fungus is a species listed in Table III.

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 μιη 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 I. 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 ofUTEX 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 (UTEXLB 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 II. 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 III. Oleaginous Fungi.

Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum,

Hensenulo, Chaetomium, Cladosporium, Malbranchea, Rhizopus, and Pythium. 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). In some embodiments, engineered microalgal cell has very low levels of polyunsaturated fatty acids. The natural oil isolated from the cell can be a liquid or solid at room temperature, or a blend of liquid and solid oils, including the

regiospecific or stererospecific oils, high stearate oils, or high mid-chain oils.

Oxidative stability can be measured by the Rancimat method using the AOCS Cd 12b-92 standard test at a defined temperature. For example, the OSI (oxidative stability index) test may be run at temperatures between 110°C and 140°C. The oil is produced by cultivating cells (e.g., any of the plastidic microbial cells mentioned above or elsewhere herein) that are genetically engineered to reduce the activity of one or more fatty acid desaturase. For example, the cells may be genetically engineered to reduce the activity of one or more fatty acyl Δ12 desaturase(s) responsible for converting oleic acid (18: 1) into linoleic acid (18:2) and/or one or more fatty acyl Δ15 desaturase(s) responsible for converting linoleic acid (18:2) into linolenic acid (18:3). Various methods may be used to inhibit the desaturase including knockout or mutation of one or more alleles of the gene encoding the desaturase in the coding or regulatory regions, inhibition of RNA transcription, or translation of the enzyme, including RNAi, siRNA, miRNA, dsRNA, antisense, and hairpin RNA techniques. Other techniques known in the art can also be used including introducing an exogenous gene that produces an inhibitory protein or other substance that is specific for the desaturase.

In a specific embodiment, fatty acid desaturase (e.g., Δ12 fatty acid desaturase) activity in the cell is reduced to such a degree that the cell is unable to be cultivated or is difficult to cultivate (e.g., the cell division rate is decreased more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97 or 99%). Achieving such conditions may involve knockout, or effective suppression of the activity of multiple gene copies (e.g. 2, 3, 4 or more) of the desaturase or their gene products. A specific embodiment includes the cultivation in cell culture of a full or partial fatty acid auxotroph with supplementation of the fatty acid or a mixture of fatty acids so as to increase the cell number, then allowing the cells to accumulate oil (e.g. to at least 40% by cell weight). Alternatively, the cells comprise a regulatable fatty acid synthesis gene that can be switched in activity. For example, the regulation can be based on environmental conditions and the environmental conditions during a first, cell division, phase favor production of the fatty acid and the environmental conditions during a second, oil accumulation, phase disfavor production of the oil.

In a specific embodiment, a cell is cultivated using a modulation of linoleic acid levels within the cell. In particular, the natural oil is produced by cultivating the cells under a first condition that is permissive to an increase in cell number due to the presence of linoleic acid and then cultivating the cells under a second condition that is characterized by linoleic acid starvation and thus is inhibitory to cell division, yet permissive of oil accumulation. For example, a seed culture of the cells may be produced in the presence of linoleic acid added to the culture medium. For example, the addition of linoleic acid to 0.25 g/L in the seed culture of a Prototheca strain deficient in linoleic acid production due to ablation of two alleles of a fatty acyl Δ12 desaturase (i.e., a linoleic auxotroph) was sufficient to support cell division to a level comparable to that of wild type cells. Optionally, the linoleic acid can then be consumed by the cells, or otherwise removed or diluted. The cells are then switched into an oil producing phase (e.g., supplying sugar under nitrogen limiting conditions as described in WO2010/063032). Surprisingly, oil production has been found to occur even in the absence of linoleic acid, as demonstrated in the obligate heterotroph oleaginous microalgae Prototheca but generally applicable to other oleaginous microalgae, microorganism, or even multicellular organisms (e.g., cultured plant cells). Under these conditions, the oil content of the cell can increase to about 10, 20, 30, 40, 50, 60, 70, 80, 90%, or more by dry cell weight, while the oil produced can have polyunsaturated fatty acid (e.g.; linoleic + linolenic) profile with 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05% or less, as a percent of total triacylglycerol fatty acids in the oil. For example, the oil content of the cell can be 50% or more by dry cell weight and the triglyceride of the oil produced less than 3% polyunsaturated fatty acids.

These oils can also be produced without the need (or reduced need) to supplement the culture with linoleic acid by using cell machinery to produce the linoleic acid, but predominantly or only during the cell division phase. The linoleic- producing cell machinery may be regulatable so as to produce substantially less linoleic acid during the oil producing phase. The regulation may be via modulation of transcription of the desaturase gene(s). For example, the majority, and preferably all, of the fatty acid Δ12 desaturase activity can be placed under a regulatable promoter regulated to express the desaturase in the cell division phase, but to be reduced or turned off during the oil accumulation phase. The regulation can be linked to a cell culture condition such as pH, and/or nitrogen level, as described in the examples herein, or other environmental condition. In practice, the condition may be manipulated by adding or removing a substance (e.g., protons via addition of acid or base) or by allowing the cells to consume a substance (e.g, nitrogen-supplying nutrients) to effect the desired switch in regulation of the destaurase activity.

Other genetic or non-genetic methods for regulating the desaturase activity can also be used. For example, an inhibitor of the desaturase can be added to the culture medium in a manner that is effective to inhibit polyunsaturated fatty acids from being produced during the oil production phase.

Using one or more of these desaturase regulation methods, it is possible to obtain a cell and/or a natural oil that it is believed has been previously unobtainable, especially in large scale cultivation in a bioreactor (e.g., more than 1000L). The oil from the cell can have polyunsaturated fatty acid levels that are 5%, 4%,

3%,2%,1%,0.5%, 0.3%, 0.2%), or less, as a percent of total triacylglycerol fatty acids in the oil.

Sterols contain from 27 to 29 carbon atoms (C27 to C29) and are found in all eukaryotes. Animals exclusively make C27 sterols as they lack the ability to further modify the C27 sterols to produce C28 and C29 sterols. Plants however are able to synthesize C28 and C29 sterols, and C28/C29 plant sterols are often referred to as phytosterols. The sterol profile of a given plant is high in C29 sterols, and the primary sterols in plants are typically the C29 sterols β -sitosterol and

stigmasterol. In contrast, the sterol profile of non-plant organisms contain greater percentages of C27 and C28 sterols. For example the sterols in fungi and in many microalgae are principally C28 sterols. The sterol profile and particularly the striking predominance of C29 sterols over C28 sterols in plants has been exploited for determining the proportion of plant and marine matter in soil samples (Huang, Wen- Yen, Meinschein W. G., "Sterols as ecological indicators"; Geochimica et

Cosmochimia Acta. Vol 43. pp 739-745).

In some embodiments the primary sterols in the microalgal biomass/oils provided herein are sterols other than β-sitosterol and stigmasterol. In some embodiments the, C29 sterols make up less than 50%, 40%, 30%, 20%, 10%, or 5% by weight of the total sterol content.

In some embodiments, the microalgal biomass/oil comprise C29 and C28 sterols, wherein the amount of C28 sterols is greater than C29 sterols. In some embodiments, the C28 sterols make up greater than 50%, 60%, 70%, 80%, 90%, or 95% by weight of the total sterol content. In some embodiments the C28 sterol is ergosterol. In some embodiments the C28 sterol is brassicasterol.

It has been found that microalgae of Trebouxiophyceae can be distinguished from vegetable oils based on their sterol profiles. Oil produced by Chlorella protothecoides was found to produce sterols that appeared to be brassicasterol, ergosterol, campesterol, stigmasterol, and β-sitosterol, when detected by GC- MS. However, it is believed that all sterols produced by Chlorella have

C24 stereochemistry. Thus, it is believed that the molecules detected as

campesterol, stigmasterol, and β-sitosterol, are actually 22,23-dihydrobrassicasterol, proferasterol and clionasterol, respectively. Thus, the biomass/oils produced by the microalgae described herein can be distinguished from plant oils by the presence of sterols with C24 stereochemistry and the absence of C24a stereochemistry in the sterols present. For example, the oils produced may contain 22,23- dihydrobrassicasterol while lacking campesterol; contain Clionasterol, while lacking in β-sitosterol, and/or contain poriferasterol while lacking stigmasterol. Alternately, or in addition, the oils may contain significant amounts of A⁷-poriferasterol.

In some embodiments, the microalgal biomass/oil comprise one or more of: at least 10%) ergosterol; ergosterol and β-sitosterol, wherein the ratio of ergosterol to β- sitosterol is greater than 25: 1; ergosterol and brassicasterol; ergosterol, brassicasterol, and poriferasterol, and wherein the oil is optionally free from one or more of β- sitosterol, campesterol, and stigmasterol.

In some embodiments, the biomass/oil provided herein comprises, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24- ethylcholest-5-en-3-ol. In some embodiments, the 24-ethylcholest-5-en-3-ol is clionasterol. In some embodiments, the biomass/oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% clionasterol.

In some embodiments, the biomass/oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24- methylcholest-5-en-3-ol. In some embodiments, the 24-methylcholest-5-en-3-ol is 22,23-dihydrobrassicasterol. In some embodiments, the biomass/oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%), or 10%) 22,23-dihydrobrassicasterol.

In some embodiments, the biomass/oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 5,22- cholestadien-24-ethyl-3-ol. In some embodiments, the 5,22-cholestadien-24-ethyl-3- ol is poriferasterol. In some embodiments, the biomass/oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% poriferasterol. In some embodiments, biomass/oil provided herein contains ergosterol or brassicasterol or a combination of the two. In some embodiments, the biomass/oil provided herein contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol. In some embodiments, the biomass/oil contains, as a percentage of total sterols, at least 25% ergosterol. In some embodiments, the biomass/oil contains, as a percentage of total sterols, at least 40% ergosterol. In some embodiments, the biomass/oil contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of a combination of ergosterol and brassicasterol.

In some embodiments, the biomass/oil contains, as a percentage of total sterols, at least 1%, 2%, 3%, 4% or 5% brassicasterol. In some embodiments, the biomass/oil contains, as a percentage of total sterols less than 10%, 9%, 8%, 7%, 6%, or 5%> brassicasterol.

In some embodiments the ratio of ergosterol to brassicasterol is at least 5: 1, 10: 1, 15: 1, or 20: 1. In some embodiments, the biomass/oil contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol and less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% β -sitosterol. In some embodiments, the biomass/oil contains, as a percentage of total sterols, at least 25% ergosterol and less than 5% β-sitosterol. In some embodiments, the biomass/oil further comprises brassicasterol. 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 lppm. 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.

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 or L*a*b coordinates. Alternatively or in addition, color characteristics may be quantified through the use of spectrophotometry or other methods known in the art.

When processed into compositions such as thermoplastics,, 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.

Delipidated biomass

After growing the cells, triglycerides may be extracted to give de-fatted biomass. Methods for oil extraction, pressing, and cell lysis are given in

WO2008/151 149, WO2010/063032, WO2010/120939, and WO2010/138620. Oil may be extracted by one or more of mechanical pressing, solvent (e.g., hexane, ethanol) extraction, enzyme treatment, 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 agent"). Where the triglyceride is produced and recovered, typically more than 5% of the dry cell weight is recovered as triglyceride. In certain cases, more than 10%>, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% of the dry cell weight may be recovered as triglyceride.

The addition of a press aid or bulking agent is advantageous in lipid extraction. When there is high oil content and low fiber in the biomass, feeding the biomass through a press can result in an emulsion. This results in low oil yields, because the oil is trapped within the solids. One method to improve the yield is to add polysaccharide to the biomass in the form of a bulking agent, also known as a "press aid" or "pressing aid". Bulking agents are typically high fiber plant polymer additives that work by adjusting the total fiber content of the microbial biomass to an optimal range. Microbial biomass such as microalgae and the like typically have very little crude fiber content. The addition of high fiber plant polymer additives (in the form of a press aid) may help adjust the total fiber content of the microbial biomass to an optimal range for oil extraction using an expeller press to prepare biomass for a particular application. Optimal fiber content for a typical oil seed may range from 10- 20%. It may be helpful to adjust the fiber content of the microbial biomass for optimal oil extraction or for a particular application. The range for fiber content in the biomass may be the same or a similar range as the optimal fiber content for a typical oil seed, although the optimal fiber content for each microbial biomass may be lower or higher than the optimal fiber content of a typical oil seed. Suitable pressing aids include, but are not limited to, corn starch, potato starch, cassava starch, switchgrass, rice straw, rice hulls, sugar beet pulp, sugar cane bagasse, soybean hulls, dry rosemary, cellulose, corn stover, delipidated (either pressed or solvent extracted) cake from soybean, canola, cottonseed, sunflower, jatropha seeds, paper pulp, waste paper and the like. In some embodiments, the spent microbial biomass of reduced lipid content from a previous press is used as a bulking agent. Thus, bulking agents, when incorporated into a biomass, change the physiochemical properties of the biomass so as to facilitate more uniform application of pressure to cells in the biomass.

Biomass processing In some embodiments, it may be desirable to further process the biomass following oil extraction. For example, the biomass may be optionally milled or ground to further reduce particle size of the biomass. The milling step may be achieved through jet milling, hammer milling, bead milling, pearl milling, or another other form of pulverization. Particle reduction may occur through roller milling. Particle reduction may occur through grinding. In some embodiments, the milled or ground biomass has a particle size of from 0.1 to 300 microns. In some embodiments, the milled or ground biomass has a particle size of from 0.1 to 10 microns, 1 to 8 microns, 2 to 7 microns, or 3 to 6 microns. In some embodiments, the milled or ground biomass has a particle size of less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micron. In some embodiments the milled or ground biomass has an average particle size about 5 microns. In some embodiments the milled or ground biomass has a particle size of from 10 to 100 microns, 100 to 200 microns, 200 to 300 microns, 300 to 400 microns or 400 to 500 microns. In some embodiments the milled or ground biomass has a particle size of from 10 to 30 microns, 30 to 50 microns, 50 to 70 microns, 70 to 90 microns, 90 to 110 microns, 110 to 150 microns, 150 to 300 microns, or 400 to 500 microns. In some embodiments, the biomass has an average particle size of greater than 50, 75, 100, 115, 125, 150, 175, 200, 225, or 250 microns (micrometer).

Biomass may be fractionated to enrich in polysaccharides or to recover proteins, nutrients or other valuable components. Fractionation may comprise washing with a solvent, especially a polar solvent such as water, ethanol or other alcohol, or mixture thereof, and centrifugation or filtration to separate soluble from insoluble fractions. Processing steps may optionally include drying or concentration to obtain biomass for use in one or more embodiments of the present invention. The drying step may be achieved through drum drying, spray drying, freeze drying, oven drying, vacuum drying, tray drying, box drying, or through another method to dry the material. Optionally, the biomass may be further milled to reduce particle size after drying or concentration.

In a further embodiment, the biomass is biodegradable or compostable. In a particular embodiment, the biomass is compostable according to ASTM D6400-04 Standard Specification for Compostable Plastics.

In some embodiments, the thermoplastic resin is polylactic acid (PLA).

Different grades of PLA are suitable for different applications or processing conditions. Non- limiting examples of PLA grades suitable for use with the microalgal biomass of this invention include NatureWorks 2002D, 2003D, 3001D, 305 ID, 3052D, 325 ID, 3801X, 4032D, 4042D, 4043D, 4050D, 4060D, 6060D, 6201D, 6201D, 6204D, 625 ID, 6252D, 6302D, 6350D, 6400D, 6752D, 7000D, 7001D, 7032D, 8052D, 825 ID, and 8302D.

The wood composites or wood plastic composites may also be blended with antioxidants. Non-limiting examples of antioxidants suitable for use with

embodiments of this invention are those such as supplied by Chemtura under the trade names ANOX^®, ULTRANOX^®, ALKNOX^®, and NAUGARD^® as well as those supplied by BASF under the trade name Iragfos^®. In an embodiment, addition of one or more antioxidant may increase the operating temperature of the composite. In a further embodiment, addition of one or more antioxidants may decrease darkening of the composite.

In an embodiment, the specific gravity of a composition prepared through blending one or more thermoplastic polymers with microbial biomass does not increase or does not significantly increase. Low or no increases in specific gravity is a desirable benefit for applications requiring light weight component parts, such as automobile components and casings for electronic equipment. In some embodiments the specific gravity of a wood plastic composite 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%), 40%), 45%), or 50%> by weight of a thermoplastic polymer is replaced with single-celled oleaginous microbial biomass, such as microalgal biomass, to form a blend.

The biomass and the composites or articles made with the biomass may be biodegradable or compostable in accordance with one or more of the following standards: ASTM D6400-04, ASTM D7071-05, ASTM D5988-03, ASTM D5511-11, ASTM D6954-04, ASTM 7475-11, ISO 1485502; 2007, ISO 14853:2005, ISO 14855-1 :2005.

In some embodiments, provided is a method for cultivating oleaginous microalgae, removing oil from the microalgae optionally using a press aid, homogenizing the delipidated biomass and producing a wood plastic composite or a wood composite from the biomass. The wood plastic or wood composite production step may use techniques disclosed here or those known in the art. For example, a wood plastic can be prepared by combining microalgal biomass with a thermoplastic polymer and a coupling agent in an intensity mixer, e.g. a ribbon blender or any low intensity mixer commonly used in blending solids. The mixture can be processed in a heated extruder at temperatures suitable for processing the particular thermoplastic polymer chosen. In another method a master batch composition can be prepared from about 50 to about 99% or about 75 to about 90%> weight of microalgal biomass and from about 1 to 5%, or about 5 to about 10 %, or about 10 to about 25% of a coupling agent or a thermoplastic resin. The resulting master batch can be blended with a thermoplastic polymer or mixture of thermoplastic polymers to obtain composites having the same range of compositions as those prepared by direct blending of the ingredients. The master batch method provides a supply that can be prepared, stored and subsequently used to react with any chosen thermoplastic polymer. Master batching also provides a method for supplying concentrated additives in pellet form. Wood composites typically comprise a formaldehyde -based resin such as urea-formaldehyde, phenol-formaldehyde or melamine-formaldehyde as the adhesive. There has been a movement away from those formaldehyde-based resins due to evidence that the off-gassed formaldehyde from the wood composite installed in a building interior may increase the risk of cancer. Methylene diphenyl diisocyanate, "MDI", is another common binder used in the production of wood composites. MDI is the most expensive component of the wood composite and therefore contributes disproportionately to the expense of the composite material. Potential health concerns related to diisocynates in conjunction with a motivation to lower overall costs are key drivers to displacing this resin in manufacture of wood composite articles.

In some embodiments, use of microalgal biomass reduces or eliminates adhesive binder in a wood composite while enabling comparable mechanical properties or other performance characteristics. In some embodiments, the biomass reduces or eliminates methylene diphenyl diisocyanate adhesives such as RUBINATE or LUPRANATE®.

In some embodiments, the biomass reduces or eliminates conventional wood plastic lubricants such as ethylene bis-stearamide (EBS), metal stearates, and lubricants supplied by Struktol Company of America under the trade name

STRUKTOL®. Accordingly the composites provided herein can contain reduced amounts of such lubricants.

In some embodiments, the combination of microalgal biomass and acid coupling agents or crosslinkers such as succinic acid, glycolic acid, glutaric acid, oxalic acid, citric acid, adipic acid, and boric acid reduces water uptake of the wood plastic composite relative to formulations lacking added said organic acids.

A group of chemicals known as sizing agents are used to hydrophobically modify the starch applied to paper in a procedure known as paper sizing. In this procedure, cellulose paper fibers are covered with a thin film of starch modified by a sizing agent, creating a water repellent surface. Reactions between starch and sizing agents have been well studied and are generally carried out at alkaline conditions and moderate temperatures. In some embodiments, microalgal biomass and alkenyl succinic anhydride, alkylated melamine, or alkyl ketene dimers may be reactively extruded with wood plastic composite formulations comprising microalgal biomass to reduce water absorbance.

EXAMPLES

Example 1: Wood plastic composite compositions comprising microalgal biomass

This example describes the use of biomass prepared from oleaginous microalgae to replace a processing lubricant in the production of wood plastic composite compositions. A genetically engineered derivative of Prototheca moriformis (UTEX 1435) was cultivated under heterotrophic conditions such as those described in WO2008/151149, WO2010/063032, and WO2011/150411, dried, then mechanically pressed to extract oil. Soybean hulls, used as a press aid in the extraction process, were added at 15% dry weight. The resulting microalgal biomass with soybean hull plant polymers retained 7.2% residual oil. The biomass

preparation, referred to as microalgal biomass Al, was milled to a final average particle size of 425 micrometers prior to compounding.

Compositions of wood plastic composites were produced comprising resin, wood flour, and optionally maleic anhydride grafted resin (MAPE or MAPP) according to the weight-based formulations given in Table IV. Zinc stearate or microalgal biomass was used as a lubricant component of the indicated formulation. Resins evaluated included high density polyethylene (HDPE, Marlex 6007, Chevron Phillips Chemical Company) and polypropylene (PP, ExonnMobil 5262). Compounds were produced using a 26mm co-rotating twin-screw extruder heated to 180°C with resin fed in the feed throat and microalgal biomass side-stuffed downstream. Flexural test bars were generated with an Engle 85 Injection Moulding Machine. Mechanical, physical, and water absorbent properties were tested according to ASTM standards (e.g. flexural strength ASTM D790, tensile strength/elongation ASTM D638, notched/unotched izod ASTM D256). Results from these tests are shown in Table V.

Table IV. Weight % formulations of materials to produce wood plastic composite compositions

Table V. Mechanical, Physical, and Water Absorbent Properties of Wood Plastic Composite Compositions prepared with microalgal biomass.

The data presented in Table V show that wood plastic composite compositions 5 prepared with microalgal biomass exhibit comparable tensile and impact properties to those prepared with added zinc stearate. The mechanical and physical properties of compositions comprising microalgal biomass Al are within 10% of tensile strength, tensile modulus, unnotched izod, and specific gravity of those of formulations made with added zinc stearate.

10 Mechanical and water resistant properties of wood plastic composite

formulations comprising microalgal biomass are improved with addition of MAPE or MAPP. Formulations comprising microalgal biomass and resin-appropriate coupling agent showed improved flexural and impact strength relative to those comprising added zinc stearate. HDPE preparations comprising microalgal biomass and MAPE showed improved water resistance compared to those prepared with added zinc stearate.

This example demonstrates the successful use of microalgal biomass to substitute for added zinc stearate to maintain or improve the mechanical and water resistant properties of wood plastic composite compositions.

Example 2: Wood plastic composite compositions comprising microalgal biomass This example describes the use of biomass prepared from oleaginous microalgae to replace different processing lubricants in the production of wood plastic composite compositions. Genetically engineered derivatives of Prototheca moriformis (UTEX 1435) were cultivated under heterotrophic conditions such as those described in WO2008/151149, WO2010/063032, and WO2011/150411, dried, then mechanically pressed to extract oil. Three different microalgal biomass samples, Bl, CI, and Dl, were prepared through pressing strains of Prototheca moriformis with soybean hulls added at the weight percentages shown in Table VI. Preparation Bl was treated with hexane to further remove residual oil. The resulting biomass preparations were milled to a final average particle size of 250-425 micrometers. Table VI. Microalgal Biomass Preparations used in compounding thermoplastic compositions

Compositions of wood plastic composites were produced comprising different amounts of high density polyethylene (HDPE, Marlex 6007, Chevron Phillips Chemical Company), recycled resin, wood flour, talc, and maleic anhydride grafted polyethylene according to the weight-based formulations given in Table VII. Zinc stearate, Struktol TPW-104, or microalgal biomass preparations were used as lubricating components of the indicated formulation. Compositions were produced using a 26 mm co-rotating twin-screw extruder heated to 180°C with resin fed in the feed throat and microalgal biomass side-stuffed downstream. Flexural test bars were generated with an Engle 85 Injection Moulding Machine. Mechanical, physical, and water absorbent properties were tested according to ASTM standards. Results from these tests are shown in Table VIII and IX.

Table VII. Weight % formulations of materials to produce wood plastic composite compositions

Table VIII. Mechanical and Water Absorbent Properties of Wood Plastic Composite Compositions prepared with microalgal biomass.

Unnotched Average 1.270 3.821 4.154 3.866 Izod (ft- lb/in),

complete St. Dev. 0.236 0.398 0.426 0.269 break

Unnotched

Izod (ft- Average 1.772 1.906 1.565 1.506

lb/in),

hinged St. Dev. 0.303 0.632 0.199 0.236

break

Specific Average 1.118 1.123 1.136 1.138 1.129 1.149 1.133 1.147 Gravity St. Dev. 0.000 0.003 0.003 0.009 0.005 0.002 0.023 0.003

% Weight

Change at Average 1.208 1.355 1.557 1.614 1.101 0.895 1.037 0.971 24 hrs

% Weight

Change at Average 1.627 2.077 2.153 2.322 1.484 1.285 1.377 1.332 48 hrs

% Weight

Change at Average 1.766 2.211 2.622 2.651 1.642 1.315 1.420 1.332 72 hrs

% Weight

Change at Average 2.015 2.411 3.085 3.203 1.685 1.550 1.577 1.561 96 hrs

% Weight

Change at Average 2.689 3.620 4.678 5.021 2.293 1.952 2.063 2.019 168 hrs

Table IX. Mechanical and Water Absorbent Properties of Wood Plastic Composite Compositions prepared with microalgal biomass.

Wood Plastic Com osite Com osition Sam le

Specific 1.120 1.143 1.122 1.145 1.267 1.244 1.305 1.306 1.309

Average

Gravity

St. Dev. 0.003 0.018 0.009 0.002 0.005 0.006 0.008 0.008 0.005

% Weight

Change at 1.445 1.192 1.130 1.172 1.406 1.781 1.453 1.528 1.293 24 hrs Average

% Weight

Change at 1.885 1.642 1.626 1.578 1.925 2.573 2.030 2.157 1.875 48 hrs Average

% Weight

Change at 2.129 1.863 1.823 1.698 2.125 2.967 2.375 2.458 1.998 72 hrs Average

% Weight

Change at 2.462 2.132 2.146 1.943 2.466 3.477 2.767 2.816 2.307 96 hrs Average

% Weight

Change at 3.538 2.989 3.132 2.863 3.588 5.300 4.127 3.975 3.414 168 hrs Average

The data presented in Table VIII show that wood plastic composite

compositions prepared with microalgal biomass types differing in oil content exhibit comparable tensile and impact properties to those prepared with added zinc stearate.

The mechanical and physical properties of compositions comprising microalgal

biomass Bl, CI, or Dl and are within 10% of tensile strength, tensile modulus,

unnotched izod, and specific gravity of those of formulations made with added zinc stearate but lacking microalgal biomass.

The data presented in Table IX show that wood plastic composite

compositions prepared with microalgal biomass exhibit comparable tensile and

impact properties to those prepared with the lubricant package Struktol TPW 104. The mechanical and physical properties of compositions comprising microalgal biomass

Dl is within 10% of tensile strength, tensile modulus, unnotched izod, and specific gravity of those of formulations made with Struktol TPW 104 but lacking microalgal biomass.

Mechanical and water resistant properties of wood plastic composite

formulations comprising microalgal biomass were improved with addition of MAPE.

Formulations comprising microalgal biomass and MAPE compatibilizer showed

improved flexural and impact strength relative to those comprising added zinc stearate and to Struktol TPW 104. HDPE preparations comprising microalgal biomass and

MAPE also exhibited improved water resistance relative to those prepared with added zinc stearate and to Struktol TPW 104.

This example demonstrates the successful use of microalgal biomass to substitute for added zinc stearate or to Struktol TPW 104 to maintain or improve the mechanical and water resistant properties of wood plastic composite compositions. Example 3: Wood plastic composite compositions comprising microalgal biomass

This example describes the use of biomass prepared from oleaginous microalgae to replace zinc stearate in the extrusion of wood plastic composite compositions. Compositions were prepared according to the sample formulations 2-1 through 2-8 listed in Table VII. Compositions were profile extruded using a miniature wood plastic composite board die setup with a Brabender profile extruder heated to 187°C. The screw speed was set at 50 rpm.

Photographs of the extruded samples 2-1 through 2-8 are shown in Figure 1. The substitution of zinc stearate with microalgal biomass generated equivalent profiles. These data demonstrate that microalgal biomass comprising different oil levels are able to replace zinc stearate in extruded wood plastic composite compositions.

Example 4: Extruded wood plastic composite compositions comprising microalgal biomass This example describes the use of microalgal biomass in the production of wood plastic composite compositions to decrease the amount of thermoplastic resin or wood flour in formulations. A genetically engineered derivative of Prototheca moriformis (UTEX 1435) was cultivated under heterotrophic conditions such as those described in WO2008/151149, WO2010/063032, and WO2011/150411, dried, then mechanically pressed with soybean hulls added at 30% by weight to extract oil.

Sucrose was used as the carbon source in the fermentation broth. The resulting microalgal biomass with soybean hull plant polymers retained 6.5% residual oil. The biomass preparation, referred to as microalgal biomass El, was milled to a final average particle size of 425 micrometers prior to compounding. Compositions of wood plastic composites were produced through combining resin, recycled resin, wood flour, lubricant, and microalgal biomass according to the weight-based formulations given in Table X then were either injection molded or extruded. Pellets for injection molded forms were produced using a 26mm co-rotating twin-screw extruder heated to 180°C with resin fed in the feed throat and microalgal biomass side-stuffed downstream. Injection molded fiexural and tensile test bars were generated with an Engle 85 Injection Moulding Machine. Mechanical and physical properties were tested according to ASTM standards. Results from these tests are shown in Table XI.

Separately, extruded profile compositions were generated upon blending and gravimetrically feeding materials into a conical twin screw Brabender profile extruder equipped with a miniature wood plastic composite board die of railroad geometry. Three zones of the extruder and the die were heated to 180°C. The screw speed was set at 100 rpm. Photographs of the extruded samples 4-1 through 4-8 are shown in Figure 2. The substitution of thermoplastic resin or wood flour with microalgal biomass generated extruded profiles that were visually equivalent to those prepared without microalgal biomass. These data successfully demonstrate that microalgal biomass is able to replace thermoplastic resin, wood flour, or combinations of resin and wood flour in formulations of extruded wood plastic composites.

Table X. Weight % formulations of materials to produce wood plastic composite compositions weight %

Microalgal Km c led Maple Struktol

Sample I I D P I :

biomass I I D P I : Hour 1 PW - 1 04

4-1 0 22.2 22.2 49.4 6.2

4-2 20 22.2 22.2 29.4 6.2

4-3 49.4 22.2 22.2 0 6.2

4-4 20 21.3 21.3 29.4 8

4-5 4 20.2 20.2 49.4 6.2

4-6 8 18.2 18.2 49.4 6.2

4-7 12 16.2 16.2 49.4 6.2

4-8 30 16 16 30 8

Table XL Mechanical Properties of Injection Molded Wood Plastic Composite Com ositions prepared with Microalgal Biomass

Example 5: Compression molded compositions comprising microalgal biomass

This example describes the use of compression molding to produce compositions with microalgal biomass. A genetically engineered derivative of Prototheca moriformis (UTEX 1435) was cultivated under heterotrophic conditions such as those described in WO2008/151149, WO2010/063032, and WO2011/150411, dried, then mechanically pressed to extract oil. Glucose was used as the carbon source in the fermentation broth. Soybean hulls, used as a press aid in the extraction process, were added at 35% dry weight. The resulting microalgal biomass with soybean hull plant polymers retained 12.2% residual oil. The biomass preparation, referred to as microalgal biomass Dl, was milled to a final average particle size of 425 micrometers prior to compounding.

Microalgal biomass Dl and different binders were combined according to the weight percentages listed in Table XII. Samples were pressed into a square plaque shape using a plate press according to the temperatures, pressures, and times listed in Table XIII. Compression molded plaques were visually evaluated for physical appearance, consistency and strength.

Table XII. Weight percent formulations used in making compression molded compositions

Table XIII. Conditions used in making compression molded compositions

The data presented in Table XIII identify combinations of binders and processing conditions that are successful in producing compression molded articles made with greater than 70% microalgal biomass.

Example 6: Thermoplastic Compositions Prepared with Biomass from

Photosynthetically and Heterotrophically Grown Microalgae

This example describes the use of biomass prepared from microalgae to produce thermoplastic compositions. Strains of microalgae were selected with differentiated levels of color generating compounds. Chlorella protothecoides (UTEX 250) and genetically engineered derivatives of Prototheca moriformis (UTEX 1435), low or lacking in chlorophyll, were cultivated under heterotrophic conditions such as those described in WO2008/151 149, WO2010/063032, and WO201 1/15041 1.

Glucose was used as the carbon source in the fermentation broth. These biomass samples were white to brown in color. Commercial samples of Chlorella vulgaris and Spirulina powders were obtained from Nuts.com (Cranford, NJ). Chlorella vulgaris and Spirulina powders were dark green in appearance due to the higher relative concentration of chlorophyll and other color generating compounds. The product information as of the date of filing assigns the Chlorella vulgaris powder to a Korean source with heterotrophic production and assigns the Spirulina powder to

photosynthetic production. Compositional analyses of the different microalgal biomass samples are presented in Table XIV.

Table XIV. Percent moisture, protein, fat, ash and carbohydrate of different microalgal biomass samples

Thermoplastic pellets were compounded using a 26mm co-rotating twin-screw extruder heated to 180°C. Marlex 6007 high density polyethylene resin was added at 60% by weight fed in the feed throat and microalgal biomass, added at 40% by weight, was side-stuffed downstream. Injection molded flexural and tensile test bars were generated from these pellets with an Engle 85 Injection Moulding Machine. Mechanical and physical properties of thermoplastic samples were tested according to ASTM standards. Results for each sample are presented in Table XV.

Table XV. Mechanical Properties of Injection Molded Thermoplastic Compositions Comprising Different Microalgal Biomass Preparations

As shown in Table XV, the mechanical and physical properties of injection molded compositions made with biomass low color compound content microalgae differ from the mechanical and physical properties of injection molded prepared from biomass from high color compound content microalgae. Specifically, biomass from microalgae comprising low levels of color compounds, relative to those cultivated photosynthetically and or with high relative levels of color compounds, results in injection molded compositions with greater impact resistance.

In addition, injection molded composites prepared with the different microalgae differ in color as evaluated by the Hunter 1948 L, a, b color space measurement. In this system, perceived lightness, L* is on a scale 0-100. a* is a measure of the hue on the red/green axis; negative values indicate green while positive values indicate magenta, b* is a measure of hue on the yellow/blue axis; negative values indicate blue while positive values indicate yellow. By the Hunter colorimeter measurement, injection molded compositions prepared with biomass from Prototheca moriformis (UTEX 1435) or Chlorella protothecoides (UTEX 250) are lighter, are shifted to farther to the red portion of the red-green axis, and are shifted farther to the blue portion of the blue-yellow axis than are injection molded compositions prepared with biomass from Chlorella vulgaris or Spirulina.

Example 7. Thermoplastic Compositions made with Microalgal Biomass prepared with different Press Aid Materials

This example describes the effects of different press aid materials used in the production of microalgal biomass to generate thermoplastic compositions with distinct mechanical properties. Genetically engineered derivatives of Prototheca moriformis (UTEX 1435) were cultivated under heterotrophic conditions such as those described in WO2008/151 149, WO2010/063032, and WO201 1/15041 1 , dried, then mechanically pressed to extract oil. Different press aids were used in mechanical extraction as described below. Glucose was used as the carbon source in the fermentation broth for biomass samples Al and Gl . Sucrose was used as the carbon source in the fermentation broth for biomass samples HI , II , and Jl .

These five different microalgal biomass samples were prepared through pressing strains of Prototheca moriformis with either soybean hulls, rice hulls, or bagasse press aid added at the weight percentages listed in Table XVI. The resulting biomass preparations were milled to a final average particle size of 250-550 micrometers.

Table XVI. Microalgal Biomass Preparations used in Compounding Thermoplastic Compositions

Thermoplastic pellets were compounded using a 26mm co-rotating twin-screw extruder heated to 180°C. Marlex 6007 high density polyethylene resin was added at 60% by weight fed in the feed throat and microalgal biomass, added at 40% by weight, was side-stuffed downstream. Injection molded flexural and tensile test bars were generated from these pellets with an Engle 85 Injection Moulding Machine. Mechanical properties of thermoplastic samples 7-1 through 7-5 were tested according to ASTM standards. Results for each sample are presented in Table XVII.

Table XVII. Mechanical Properties of Injection Molded Thermoplastic Compositions Com rising Different Microalgal Biomass Preparations

The data presented in Table XVII show that the use of different press aid materials used in preparation of the microalgal biomass can be varied to improve tensile, flexural, or impact properties of injection molded compositions prepared with microalgal biomass.

The result of pressing microalgae with soybean hulls to extract oil, in contrast to pressing with rice hulls or bagasse, is a microalgal biomass that imparts greater elongation and impact resistance to a thermoplastic composition. Among the samples listed in Table XVII, materials produced from microalgal biomass pressed with soybean hulls have the greatest elongation, notched Izod, and unnotched Izod values. The result of pressing microalgae with rice hulls or bagasse to extract oil, in contrast to pressing with soybean hulls, is a microalgal biomass that imparts greater tensile and flexural strength and modulus to a thermoplastic composition. Among the samples listed in Table XVII, materials produced from microalgal biomass pressed with bagasse have the highest tensile and flexural strength and modulus. This example demonstrates that distinct press aid materials were used to improve the mechanical properties of injection molded thermoplastic compositions comprising microalgal biomass.

Example 8: Injection molded compositions comprising triglyceride oil and microalgal biomass This example describes the use of triglyceride oil and microalgal biomass and to produce thermoplastic compositions with distinct mechanical properties. A genetically engineered derivative of Prototheca moriformis (UTEX 1435) was cultivated under heterotrophic conditions such as those described in WO2008/151149, WO2010/063032, and WO2011/150411, dried, then mechanically pressed to extract oil. Sucrose was used as the carbon source in the fermentation broth. Microalgal biomass sample El was prepared through pressing Prototheca moriformis with soybean hulls added at 30% by weight. The resulting microalgal biomass with soybean hull plant polymers retained 6.5% residual oil. The resulting biomass preparation was milled to a final average particle size of 400 micrometers. Where indicated, triglyceride oil was added, then mixed with microalgal biomass sample El to increase the amount of oil in the microalgal biomass.

Thermoplastic pellets of the weight-based formulations corresponding to the samples in Table XVIII were compounded using a 26mm co-rotating twin-screw extruder heated to 180°C with resin fed in the feed throat and microalgal biomass side-stuffed downstream. Injection molded test bars were generated from these pellets with an Engle 85 Injection Moulding Machine. Mechanical properties were tested according to ASTM standards. Results for each sample are presented in Table XIX. Table XVIII. Microalgal Biomass formulations used in compounding thermoplastic compositions

Table XIX. Mechanical Properties of Injection Molded Thermoplastic Compositions 5 Com rising Microalgal Biomass

The data presented in Table XIX show that the mechanical properties of injection molded compositions prepared with microalgal biomass can be altered through increasing the oil content of the microalgal biomass.

The result of adding triglyceride oil to microbial biomass sample El prior to compounding and extrusion was an increase in the elongation and in the impact resistance of injection molded parts made with the compounded microalgal biomass. In high density polyethylene, elongation improved from about 10% (Sample 8-1) to about 13% (Sample 8-3) as a result of mixing additional triglyceride oil into microalgal biomass El . In polypropylene, elongation improved from about 3% (Sample 8-5) to about 5% (Sample 8-8) as a result of mixing additional triglyceride oil into microalgal biomass El . In high density polyethylene, notched Izod improved from about 1.445 ft-lb/in (Sample 8-1) to about 1.603 ft-lb/in (Sample 8-4) and unnotched Izod improved from about 2.117 ft-lb/in (Sample 8-1) to about 3.223 ft- lb/in (Sample 8-4) as a result of mixing additional triglyceride oil into microalgal biomass El . In polypropylene, unnotched Izod improved from about 0.953 ft-lb/in (Sample 8-5) to about 1.625 ft-lb/in (Sample 8-8) as a result of mixing additional triglyceride oil into microalgal biomass El .

This example demonstrates the successful use of added triglyceride oil to microalgal biomass to improve the mechanical properties of injection molded thermoplastic compositions.

Example 9:

This example describes the detection of volatile compounds from

thermoplastic compositions prepared with different microalgal biomass samples. Injection molded tensile bars were produced according the weight percentages listed in Table XX using the methods outlined in Examples 6, 7, and 8. The microalgal biomass samples utilized in generating these compositions were described in Examples 4 through 8.

Table XX. Weight percent formulations

9-7 60 40 HI

9-8 60 40 Jl

The different tensile bar samples were characterized by different odors and aromas. Sample 9-2 was characterized as having a musty, earthy, damp, and fishy smell. Sample 9-3 was characterized by grassy, barny, smoky, and seaweed odors. In contrast, Samples 9-4 through 9-8 were characterized by malty, sweet, toasty, caramel, burnt popcorn, and cotton candy odors.

Each tensile bar was subjected to volatile analysis through a process of extraction and detection by GC-MSD. All injections were splitless. Peak identification was based on comparison of EI mass spectra in samples to EI mass spectra of the NIST Library. Data reported are as relative concentration compared to the internal standard expressed in ppb.

The total list of detected compounds across Samples 9-1 through 9-8 was evaluated for those uniquely present in thermoplastic samples prepared with Chlorella vulgaris or Spirulina biomass (Table XXI) and for those uniquely present in thermoplastic samples prepared with Prototheca moriformis (UTEX 1435) biomass (Table XXII). Compounds listed in Table 10X were not detected from thermoplastic samples prepared with Prototheca moriformis (UTEX 1435) biomass. The minimum and maximum detected levels reported in the tables below are in units of parts per billion, determined relative to a 2-undecanone internal standard. CAS numbers for the compounds are listed.

Table XXL Compounds detected from injection molded compositions prepared with Chlorella vulgaris or Spirulina biomass

Thiazole, 2,4-dimethyl- 541-58-2 7.9 30

1 -Butylpyrrolidine 767-10-2 18.4 85.6

Butanal, 2-ethyl- 97-96-1 21.8 50.6

Pyrazine, 2,5-dimethyl- 123-32-0 831.5 1224

Pyrazine, ethyl- 13925-00-3 17.1 161.9

Pyrazine, 2,3-dimethyl- 5910-89-4 67.6 152.1

Pyridine, 2,4-dimethyl- 108-47-4 15 67.4 l-Octen-3-ol 3391-86-4 14.1 181.2

5-Hepten-2-one, 6-methyl- 110-93-0 100.6 120.7

2-Octanone 111-13-7 32.3 188.9

Pyrazine, trimethyl- 14667-55-1 530 853

2-Cyclohexen- 1 -one, 3 ,5 ,5 -trimethyl- 78-59-1 39.9 425.3

Pyrazine, 2-ethyl-3,5-dimethyl- 18138-04-0 132.7 443.4

2-Cyclohexen- 1 -one, 3 ,5 ,5 -trimethyl- 78-59-1 11.7 12.1

Pyrazine, 2,3-diethyl-5-methyl- 18138-04-0 1.5 84.7

Pyrazine, 3,5-diethyl-2-methyl- 18138-05-1 98.1 102.7

Pyrazine, 2-methyl-5-(l-propenyl)-, (E)- 18217-82-8 54.1 275.2

1 -Cyclohexene- 1 -carboxaldehyde, 2,6,6- trimethyl- 432-25-7 42.1 230

Octadecane, 6-methyl- #N/A 35.7 40.3

Ionone 127-41-3 44 338.6

Geranyl acetone 3796-70-1 356.8 693.7

2(4H)-Benzofuranone, 5,6,7,7a- tetrahydro-4,4,7a-trimethyl- 17092-92-1 55 1379

Table XXII. Compounds detected from injection molded compositions prepared with Prototheca moriformis (UTEX 1435) biomass

HMF 67-47-0 967.1 3662.3

Nonanoic acid 112-05-0 57.8 131.3

2',6'-Dihydroxyacetophenone 699-83-2 48.7 1451.7

5 - Acetoxymethyl-2-furaldehyde 10551-58-3 64.4 190.1 n-Decanoic acid 334-48-5 7.6 171.8

4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6- methyl- 28564-83-2 Nd 9571.0

Thermoplastic compositions prepared with microalgal biomass from Chlorella vulgaris or Spirulina are characterized by nitrogenous compounds such as pyridines, pyrazines, pyrroles, and pyrrolidines. Thermoplastic compositions prepared with microalgal biomass from Prototheca moriformis (UTEX 1435) lack these nitrogenous compounds and are, in contrast, characterized by furan and alcohol compounds.

Example 10: Injection molded polypropylene compositions comprising glass fiber and microalgal biomass

This example describes the use of microalgal biomass and glass fiber and to produce thermoplastic compositions with desirable mechanical properties.

Thermoplastic pellets of formulations corresponding to the samples in Table XXIII were compounded using a 26mm co-rotating twin-screw extruder heated to 180°C with 35G-00 polypropylene homopolymer (INEOS Olefins & Polymers USA) fed in the feed throat and microalgal biomass and Thermo flow 738 4mm glass fiber side- stuffed downstream. Microalgal biomass D 1 , described in Example 5 , was used in these thermoplastic samples. Injection molded flexural and tensile test bars were generated from these pellets with an Engle 85 Injection Moulding Machine.

Mechanical properties were tested according to ASTM standards. Results for each sample are presented in Table XXIV. Table XXIII. Weight percent formulations

Table XXIV. Mechanical and Physical Properties of Injection Molded Thermoplastic Com ositions Comprising Microalgal Biomass and Glass Fiber

The data presented in Table XIV show that the flexural and tensile modulus of glass fiber filled injection molded compositions prepared with microalgal biomass are comparable or improved relative to those of injection molded parts lacking microalgal biomass.

This example demonstrates the successful use of microalgal biomass to lower the amount of thermoplastic resin in a glass filled composite while improving specific mechanical properties.

Example 11: Impact strength of thermoplastic compositions prepared with microalgal biomass

This example describes the use of biomass prepared from microalgae to produce thermoplastic compositions with improved impact strength. Prototheca moriformis (UTEX 1435) was cultivated under heterotrophic conditions such as those described in WO2008/151 149, WO2010/063032, and WO201 1/15041 1 , dried, then mechanically pressed to extract oil. Dl and Fl microalgal biomass preparations were obtained through alterations in processing, oil extraction, and milling conditions. Preparation Dl was described in Example 5. Sucrose was used as the carbon source in 5 the fermentation through which Preparation Fl was generated; following drying

microalgal biomass was mechanically pressed with soybean hulls added at 30% by weight to extract oil. Fl was characterized by 9% residual oil.

Dl and Fl were milled to a final average particle size of 250-425 micrometers then compounded with polypropylene copolymer (ExxonMobil PP7033N), 0.25% by

10 weight antioxidant, 2% by weight coupling agent, and 10% by weight elastomer.

Weight percentages of microalgal biomass and polypropylene copolymer are shown in Table XXV. Compounding was conducted with a 26mm co-rotating twin-screw extruder with resin fed in the feed throat and microalgal biomass side-stuffed downstream. Injection molded test bars were generated with an Engle 85 Injection

15 Moulding Machine. Mechanical properties of the compositions were tested according to ASTM standards. Results from these tests are shown in Table XXV.

Table XXV. Formulations and Mechanical Properties of Injection Molded

Compositions

11-10 40 47.75 1.475 0.096 4.788 1.529

As shown in Table XXV, different microalgal biomass preparations are associated with different thermoplastic composition mechanical properties.

Formulations with 30% Dl and 57.75% polypropylene copolymer were characterized by a Notched Izod of about 2.6 ft-lb/in. Formulations with 20% Dl and 67.75% polypropylene copolymer were characterized by a Notched Izod of greater than about 3.2 ft-lb/in.

Example 12. Melt Flow Indexes

This example describes the use of biomass prepared from microalgae to produce thermoplastic compositions with desired impact strength in polypropylene copolymers (PPcPs) that differ in melt flow rates. Microalgal biomass Dl, described in Example 5, was blended and extruded with polypropylene copolymer, 0.25%> by weight antioxidant, 2% by weight coupling agent, and 10%> by weight elastomer as described in Example 11. Three distinct polypropylene copolymers differing in molecular weight and as a result, differing in melt flow index (MFI), were

compounded with microalgal biomass Dl according to the weight percentage

formulations listed in Table XXVI. ExxonMobil PP7033N had an MFI of 8 g/10 minutes, Lyondell Basell Profax SG702PP had an MFI of 18 g/10 minutes, and

Lyondell Basell SG899 PP had an MFI of 35 g/10 minutes. Compounding was conducted with a 26mm co-rotating twin-screw extruder with resin fed in the feed throat and microalgal biomass side-stuffed downstream. Injection molded test bars were generated with an Engle 85 Injection Moulding Machine. Mechanical properties of the compositions were tested according to ASTM standards. Results from these tests are shown in Table XXVI. Table XXVI. Formulations and Mechanical Properties of Injection Molded

Compositions

12-4 30 57.75 18 2.5598 0.157 6.5162 1.053

12-5 20 67.75 35 3.8982 0.368 9.542 0.804

12-6 30 57.75 35 2.589 0.297 6.4234 0.697

As shown in Table XXVI, the formulations with 30% Dl and 57.75%

polypropylene copolymer were characterized by a Notched Izod value of about 2.6 ft- lb/in. Formulations with 20% Dl and 67.75% polypropylene copolymer were

characterized by a Notched Izod value of greater than about 3.2 ft- lb/in. These results demonstrate that injection molded compositions exhibiting desired impact strength properties may be produced with microalgal biomass and distinct molecular weight polypropylene copolymers.

Example 13: Masterbatch Compositions Prepared with Microalgal Biomass This example describes the production of masterbatch compositions prepared with microalgal biomass to produce injection molded thermoplastic articles with desired mechanical and physical properties. Masterbatch compositions of microalgal biomass Dl (described in Example 5), polypropylene copolymer, antioxidant, coupling agent, and elastomer were compounded with a twin screw according to the weight-based formulations listed in Table XXVII.

The pelletized masterbatch sample described in Table XXVII was then let down by injection molding with added polypropylene copolymer pellets to match the weight based formulations listed in Example 12, Table XXVI. Specifically, 45% by weight masterbatch sample was combined with 55% by weight of the three different polypropylene copolymer resins listed in Table XXVI. Injection molded test bars were generated with an Engle 85 Injection Moulding Machine. Mechanical properties of the compositions were tested according to ASTM standards. Results from these tests are shown in Table XXVIII.

Table XXVIII. Formulations and Mechanical Properties of Injection Molded Compositions

As shown in Table XXVIII, injection molded forms produced from microalgal masterbatch and different molecular weight polypropylene copolymers were characterized by Notched Izod values of about 2.85, 2.95, and 3.04 ft-lb/in. These results demonstrate that injection molded compositions exhibiting desired impact strength properties were produced with masterbatch compositions prepared with high concentrated levels of microalgal biomass.

Example 14: Masterbatch Compositions Prepared with Microalgal Biomass This example describes the production of masterbatch compositions prepared with microalgal biomass to produce injection molded thermoplastic articles and wherein the physical and mechanical properties of the compositions prepared through masterbatch are comparable to those produced through direct formulation

compounding and injection molding. Masterbatch compositions of microalgal biomass Dl (described in Example 5) and C I (described in Example 2) and high density polyethylene (Marlex 6007) were compounded with a twin screw according to the weight-based formulations listed in Table XXIX.

Table XXIX. Weight Percent Formulation of Masterbatch Pellets

Microalgal % M icroalgal %

Master!). itch

Biomass Bi in ass I I DIM:

13-1 Dl 80 20

13-2 C I 80 20

The pelletized masterbatch samples were then let down by injection molding with added high density polyethylene pellets to match the weight based formulations listed in Example 8, Table XVIII. Specifically, 50% by weight masterbatch sample was combined with 50%> by weight of HDPE pellets. Injection molded test bars were generated with an Engle 85 Injection Moulding Machine. Mechanical properties of the compositions were tested according to ASTM standards. Results from these tests are shown in Table XXX.

Table XXX. Formulations and Mechanical Properties of Injection Molded

Compositions

These results demonstrate that injection molded compositions may be prepared through a masterbatch format, comprising microalgal biomass at 80% weight, to achieve comparable mechanical performance to that of injection molded compositions formulated directly with lower levels of microalgal biomass.

Example 15: Microalgal Biomass- Wood Composite Formulations

This example describes the production of biomass and wood composite formulations to produce pressed biomass-wood composite panels in different material proportions. Materials include wood fiber, methylene diphenyl diisocynate (MDI), algal biomass containing press-aid, algal biomass absent press-aid, and ethanol extracted algal biomass. The wood fiber was pulverized, fluffed, and sieved to create a consistent particle size. The wood fiber was mixed with 2% water to bring the final moisture content to approximately 8% to enhance the MDI binding reaction. A pan coater was used to spray the MDI onto tumbled wood fiber which was subsequently mixed with biomass and mixed for an additional 5 minutes. The resulting mixture underwent an initial press to create a 2 inch cake. The 2 inch cake was mechanically pressed at 250°F and 600psi then cooled with refrigerant to 100-1 10°F creating a 1/2 inch composite board. Table XXXI. Microalgal Biomass- Wood Composite Formulations

Control panels with varying proportions of wood fiber, biomass, and methylene diphenyl diisocyanate (MDI) created baseline performance standards for conventional composite formulations. Controls 1 and 2 comprised a mixture of MDI and wood fiber in varying proportions. Control 3 was comprised of biomass made from a mixture of algae and soy hulls mixed with MDI and wood fiber.

The experimental composites contained a range of 2-3% MDI, 73-88% wood fiber, and 10-25% algal biomass. Unlike the biomass source contained in Control 3, experimental composites were created from biomass from purely algal sources resulting from ethanol extraction or press extraction without press-aid. In total, the results show 9 different biomass-wood composite formulations including 3 control composites and 6 experimental composites. Example 16: Wood-Plastic Composites and Microalgal Biomass Formulation

Wood plastics composites were prepared in 28 compositions, each with varying ratios of coupling agents (Polybond 3029, a maleic anhydride grafted HDPE polymer), lubricants (TPW 113), wood fiber, HDPE, talc, and biomass. The composites were subsequently extruded at 300-350° F to create samples of the 28 compositions. The various formulations of wood-plastics composites were left for a period of 7 days to examine water uptake with measurements taken after 24 hours and at 7 days.

Table XXXII. Wood-Plastic Composites and Microalgal Biomass Formulations

These results demonstrate that wood-plastic composites (WPC) combined with biomass can produce composite materials that behave similarly to conventional WPCs in water uptake performance. Notably, WPC-biomass formulations 1, 9, 11, and 19 behaved most similarly to the wood-plastic control composites. In some cases, the WPC-biomass formulations absorbed less water than Control 1. Water uptake performance comparable to the controls was achieved when WPC-biomass composites did not exceed 5% biomass content.

Example 17: Microalgal Biomass Char Proximate Ultimate Energy Analysis

Biomass samples prepared according to the examples provided above were subjected to proximate energy analysis to determine the molecular compositions produced by the samples after torrefaction. Samples were analyzed to determine the moisture, crude protein, crude fat, fiber, ash, volatiles, fixed carbon, and percent yield of char after torrefaction. Torrefied sample 1 contained less moisture, crude fat, and volatiles after torrefaction than the control samples as shown in Tables XXXIIIa-b.

Table XXXIIIa. Microalgal Biomass Char Proximate Ultimate Energy Data

Table XXXIIIb. Microalgal Biomass Char Proximate Ultimate Energy Data

Example 18: Additive compositions that improve water resistance of wood plastic composites

This example demonstrates the effectiveness of various coupling agents to decrease the water uptake of wood plastic composite compositions comprising microalgal biomass. Microalgal biomass was prepared according to the examples provided above and milled to a final average particle size of 250-425 micrometers. Premixes of wood flour, microalgal biomass, low density polyethylene (Plascoat, Surrey UK), coupling agents, and other additives were combined according to the weight percentages shown in Table XXXIV and mixed for 1 hour by tumbling in a ball jar. Compounding of premixes was conducted with a DSM Xplore mini-compounder twin conical screw 5 Hot Melt Extruder (HME) integrated to an injection moulder (15mL barrel with co- rotating screws). Compounding was performed at a screw speed of 40 rpm, and a melt temperature of 200°C. Typical mix time is approximately 20 minutes (barrel recirculates until the mixing force, as measured via screw force, equilibrates).

Injection molded dogbone test bars were formed with DSM Mould #UX16. Dogbone

10 test articles were marked, weighed, and evaluated for water absorption following 24 and 168 hr immersion in deionized water. At the time duration indicated, dogbones were removed from the water, patted dry with a Kimwipe™ and set in room temperature air for 30 minutes. The articles were then re-weighed and the mass of absorbed water calculated as the difference between the initial and timepoint masses.

15 Water absorption results are reflective of {[(initial mass) - (timepoint mass)]/(initial mass)} x 100%. Results from these tests are shown in Table XXXIV.

Table XXXIV. Weight % formulations of materials to produce wood plastic composite compositions and water absorption of compounded articles

50 44 5 0 0 0 1.0 0 0 2.25 6.33

50 42.5 5 0 0 0 0 2.5 0 2.21 5.95

50 42.5 5 0 0 0 0 0 2.5 1.17 3.78

The data presented in Table XXXIV show that the water absorption of wood plastic composites comprising microalgal biomass may be reduced with inclusion of different organic acids into the wood plastic composite formulation. The result of inclusion of 0.5%, 1.0%>, or 2.0% succinic acid into a wood plastic composite premix is an improvement of approximately 18%, 20%, and 25% in water absorption relative to samples prepared without succinic acid. The result of inclusion of 2.0% glutaric acid into a wood plastic composite premix is an improvement of approximately 25% in water absorption relative to samples prepared without glutaric acid. The result of inclusion of 2.0% glycolic acid into a wood plastic composite premix is an

improvement of approximately 25% water absorption relative to samples prepared without glycolic acid. The result of inclusion of 2.5% oxalic acid into a wood plastic composite premix is an improvement of approximately 30% water absorption relative to samples prepared without oxalic acid. Example 19: Additive compositions that improve water resistance of wood plastic composites

This example demonstrates the effectiveness of various organic acid coupling agents to decrease the water uptake of wood plastic composite compositions

comprising increased microalgal biomass, while concomitantly reducing or

eliminating costly lubricant additives.

Microalgal biomass was prepared according to the examples provided above and milled to a final average particle size of 250-425 micrometers. Premixes of wood flour, microalgal biomass, powdered high density polyethylene, coupling agents, talc, and other additives were combined according to the weight percentages shown in

Table XXXV. Compounding of premixes, injection molding of samples, and

evaluation of water absorption was conducted as outlined in Example 18. The

extrusion force used in preparing samples is shown in Table XXXVI.

Table XXXV. Weight % formulations of materials to produce wood plastic

composite compositions and water absorption of compounded articles

Table XXXVI. Extrusion force used in Compounding Articles

The data presented in Table XXXV show that the water absorption of wood plastic composites comprising microalgal biomass may be reduced with inclusion of different organic acids into the formulation. The result of inclusion of 2.0% succinic acid or 2.0% glycolic acid into a wood plastic composite formulation with 10% microalgal biomass is a decrease of approximately 40% in water absorption relative to a similar sample prepared without succinic acid or glycolic acid.

The data presented in Table XXXVI show that organic acid coupling agents can be added to wood plastic composite formulations comprising microalgal biomass lacking lubricant to decrease the extrusion force used in compounding relative to formulations compounded without organic acid coupling agents. The result of inclusion of 2.0% succinic acid or 2.0% glycolic acid into a wood plastic composite formulation with 10% microalgal biomass is a decrease of 8% or 15% compounding force relative to similar formulations compounded without organic acid coupling agents.

Example 20: Microalgal biomass treated with 1% Alkylated Melamine

Into a 600 ml beaker was placed 2 grams of BERSIZE®6549 Surface Size (a 50% aqueous emulsion of alkylated melamine from Bercen, Denham Springs, LA) and 250 ml of deionized water. 99 grams of microalgal biomass, prepared as indicated in the above experiments, were added to this aqueous emulsion and hand mixed well with a spatula. The mixture was then mixed in a high-shear blender (Silverson Mixer) for 8 minutes at 8000 rpm. The resulting mixture exhibited an increase in temperature to 50°C. It was transferred to a pan, spread evenly, and then placed in a vacuum oven and heated at 110°C under -760mm Hg pressure until dry. The dried cake was brittle and easily broke into small pieces. The final product was 1% Alkylated Melamine.

The above alkylated melamine-treated microalgal biomass (0.5 grams) was placed in a test tube with 10 g deionized water and mixed. Upon shaking the tube an immediate settling of the solids was noted with clear tan liquid above. In contrast, mixing of 0.5 g unmodified microalgal biomass to 10 g deionized water resulted an opaque tan liquid. The treatment process of microalgal biomass with alkylated melamine appears to have changed the properties upon water exposure.

Example 21: Microalgal biomass treated with 1% Alkenyl Succinic Anhydride (ASA)

Into a 600 ml beaker was placed 1 gram of BERSIZE®7920 Synthetic Internal

Size (an octadecenyl succinic anhydride with approximately 96.7% CI 8 and 3.3% C16 succinic anhydride from Bercen, Denham Springs, LA) and 250 ml of deionized water. 99 grams of microalgal biomass, prepared as indicated in the above

experiments, were added to this aqueous emulsion and hand mixed well with a spatula. The mixture was then mixed in a high-shear blender (Silverson Mixer) for 8 minutes at 8000 rpm. The resulting mixture exhibited an increase in temperature to 47°C. The mixture was transferred to a pan, spread evenly, and then placed in a vacuum oven and heated to 80°C- 110°C under -760mm Hg pressure until dry. The dried cake was brittle and easily broke into small pieces. The final product is 1% ASA.

The above ASA-treated microalgal biomass (0.5 grams) was placed in a test tube with 10 grams deionized water and mixed. Upon shaking the tube with the ASA- treated AMP, an immediate settling of the solids was noted with clear tan liquid above. The treatment process of microalgal biomass with the ASA appears to have changed the properties upon water exposure.

Example 22: Microalgal biomass treated with 10% or 20% Alkenyl Succinic Anhydride (ASA)

The process outlined in Example 22 was repeated but with an increase in the ASA loading to either 10% or 20%>. The resulting dried cakes were rubbery in texture, with the sample with 20% ASA exhibiting increased flexibility. The final products were either 10% or 20% ASA. Example 23: Microalgal biomass Treatment with 2% Alkyl Ketene Dimer (AKD)

Into a 600 ml beaker was placed 1 gram of EKA DR C222 Alkyl Ketene Dimer (a 15% aqueous dispersion from Akzo Nobel, Marietta, GA) and 150 ml deionized water. Microalgal biomass, 49 grams prepared as indicated in the above experiments, was added to this aqueous emulsion and hand mixed well with a spatula. The mixture was transferred to a pan, spread evenly, and then placed in a vacuum oven and heated to 65°C until dry. The dried cake was brittle. The material was broken into smaller pieces by hand. The final product is 2%.

Example 24: Wood Composite Articles comprising Microalgal biomass

An appropriate amount of MDF (medium density fiberboard) pine wood fiber (sufficient to enable good application of MDI onto fiber) was added into a rotary drum mixer. Methylene diisocyanate (MDI, Rubinate 1840 from Huntsman) and sufficient water to bring the moisture content of the resulting resinated fiber to ~10 wt% were sprayed onto the MDF fiber while it was tumble mixed in a rotary drum mixer. The resinated MDF fiber was tumble mixed for ~10 minutes after spraying completion. The resinated MDF fiber was removed from the rotary drum mixer and fiber clumps were "opened" using a knife-blade mill. The appropriate amount of resinated MDF fiber and press-aid containing microalgal biomass were combined and tumble mixed using the rotary drum mixer. A pre-weighed sample of this mixture was taken for analysis of moisture content by weight loss after drying using a warm air dryer. The resinated MDF fiber/ AMP mixture was placed into a wooden preform, about 24 inch x 24 inch on top of a release sheet on a metal plate and then spread to a uniform depth. A wooden panel was placed on top of the resinated MDF fiber/bioproduct mat and hand pressed down to form a partially compressed mat. The wooden preform and panel were removed, another release sheet placed on top of the mat followed by a metal plate, and this entire assembly placed into a press pre-heated to the desired compression temperature, 250°F. Press parameters were set to result in a 1/8 inch final panel thickness with press times between 22 to 26 minutes, followed by cooling to ~110°F. Typical maximum pressures were approximately lOOOpsi.

Cooling of the panel at pre-set platen gap is required to enable entrapped volatiles to slowly escape from the panel, reducing the opportunity for void formation within the panel causing an excessive increase in panel thickness upon press opening. After opening the top metal plate and release sheet were removed, and the resulting panel removed from the press and allowed to cool to ambient temperatures.

A strip around the panel edge strip was cut off such that a panel of uniform thickness was obtained. Both the dimensions and weight of this panel were measured and used to determine the panel density. Test articles were then cut from the panels and their Modulus of Elasticity (MOE, modulus in table), Modulus of Rupture (MOR, rupture in Table), and InterBond (IB) Strength measured according to ASTM methods. Water uptake was determined by comparing initial thickness, length, width and weight to the measurements following a 2 hour immersion and a 24 hour immersion with no drying period. Wood composite compositions and test results are given in Tables XXXVII through XXXIX.

Table XXXVII. Wood Composite Compositions

Table XXXVIII. Wood Composite Properties and Mechanical Results

(Ib/ft3) (in) (in) (psi) (psi) (Ibf) (Ibf) (psi)

1-1 52.5 2.005 0.115 197000 1050 6 257.6 64.4

1-2 55.9 2.000 0.111 258000 1630 9 193.6 48.4

1-3 62.0 2.004 0.107 395000 2710 14 280.7 70.2

Average 56.8 2.003 0.111 283000 1800 9.7 244.0 61.0

COV 8.46% 0.14% 3.52% 35.83% 46.8% 40.18% 18.50% 18.50%

2-1 62.5 2.006 0.092 398000 3100 12 368.5 92.1

2-2 68.9 2.010 0.094 575000 3940 16 166.3 41.6

2-3 62.1 2.008 0.094 384000 2690 11 186.8 46.7

Average 64.6 2.008 0.093 452000 3240 12.6 240.5 60.1

COV 5.86% 0.09% 1.50% 19.67% 19.67% 20.95% 46.27% 46.27%

3-1 46.8 2.009 0.123 327000 3470 23 325.3 81.3

3-2 46.4 2.005 0.131 291000 2870 22 332 83.0

3-3 47.8 2.007 0.131 349000 3230 25 288.5 72.1

Average 47.0 2.007 0.128 322000 3190 23.3 315.3 78.8

COV 1.49% 0.10% 3.53% 9.09% 9.47% 5.70% 7.43% 7.43%

4-1 60.8 2.005 0.097 490000 4650 19 365.5 91.4

4-2 60.9 2.001 0.102 540000 4910 23 519.5 129.9

4-3 56.9 2.000 0.110 461000 4330 23 523 130.8

Average 59.5 2.002 0.103 497000 4630 21.9 469.3 117.3

COV 3.79% 0.13% 6.26% 8.04% 6.27% 9.40% 19.16% 19.16%

Table XXXIX. Wood Composite Properties and Mechanical Results

Example 25: Wood Composites Comprising Eucalyptus Fiber and Microalgal Biomass

Composite panels were prepared comprising microalgal biomass, eucalyptus sawdust, and MDI binder. A 5in x 5-1/8 in square deckle box was set onto a sheet of quick release aluminum foil laid on a metal plate. Microalgal biomass prepared without press aid, eucalyptus sawdust and Rubinate® MI resin were combined and mixed using a rotary drum mixer according to the weight percentages indicated in Table XL. The admixture was then added to the prepared deckle box up to a measured depth. The mixture was leveled with a wooden board and then pre-pressed with a plunger. After the deckle box and plunger were removed, l/8in. thick metal nuts were placed at the corners of the square layer of the bioproduct and sawdust mixture. The layer was topped with a sheet of quick release aluminum foil and a second metal plate. The sandwiched material was put into a press at 250°F for 20 minutes and treated to forming pressures of about 5,000 psi, about 10,000 psi, or about 20,000 psi then cooled to 100°F before removal from the press. The finished panels were measured and weighed to determine density. Water uptake and stress strain parameters were determined as outlined in Example 24.

Compositions, thickness and density, strength, and water uptake of resulting panels are given in Tables XL-XLI. Table XL. Eucalyptus Wood Composite Properties and Mechanical Results

Table XLI. Eucalyptus Wood Composite Water Uptake Properties

The described embodiments provided herein are intended to be merely exemplary and numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A wood plastic composite comprising a blend of:

a) a thermoplastic resin;

b) a cellulosic filler;

c) an oleaginous microbial biomass, and

d) a binder or a coupling agent,

wherein the biomass is optionally thermochemically treated.

2. The composite of claim 1, wherein the thermoplastic resin is selected from the group consisting of a polystyrene, polyolefm, polyvinyl chloride, polylactic acid, and polymethyl methacrylate resin.

3. The composite of claim 2, wherein the polyolefm is recycled.

4. The composite of claim 2 or 3, wherein the polyolefm is polyethylene or polypropylene.

5. The composite of claim 4, wherein the polyethylene is low density

polyethylene (LDPE), high density polyethylene (HDPE), or recycled HDPE.

6. The composite of any one claims 1 to 5, wherein the thermoplastic resin constitutes up to 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85% by weight of the composite.

7. The composite of any one of claims 1 to 6, wherein the coupling agent is a silane, an organic acid, a di-acid, a tri-acid, an anhydride, a cyclic anhydride, boric acid, a maleic anhydride grafted polyolefm, succinic acid, succinic anhydride, glutaric acid, glycolic acid, oxalic acid, citric acid, or adipic acid.

8. The composite of claim 7, wherein the coupling agent is maleic anhydride grafted high density polyethylene (MAPE) or maleic anhydride grafted polypropylene (MAPP).

9. A wood composite comprising a blend of:

a) a cellulosic filler;

b) an oleaginous microbial biomass, and c) a binder;

wherein the wood composite is not a wood plastic composite.

10. The composite of claim 9, wherein the binder is urea formaldehyde, phenol formaldehyde, melamine, an isocyanate, polymeric diphenyl methane diisocyanate (polymeric MDI), an emulsion polymer isocyanate, resorcinol, phenol resorcinol, or an epoxy resin.

11. The composite of claim 10 wherein the binder is an isocyanate, methylene diphenyl diisocyanate (MDI), or polymeric methylene diphenyl diisocyanate.

12. The composite of claim 9, wherein the binder is polyvinyl acetate, a polyurethane, a polyurethane/emulsion polymer, an elastomer, a hot-melt, starch, casein, blood, or animal glue.

13. The composite of any one of the preceding claims, wherein the binder or coupling agent is present in an amount of up to 0.25, 0.5, 1, 2, 3, 4, or 5% by weight of the composite.

14. The composite of any one of the preceding claims, wherein the oleaginous microbial biomass is delipidated.

15. The composite of claim 14, wherein the delipidated biomass comprises residual lipid that is present in an amount of up to 20%, 19%>, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% by weight of the biomass.

16. The composite claim 14 or 15, wherein the oleaginous microbial biomass is delipidated such as by pressing or solvent extraction.

17. The composite of claim 16, wherein the oleaginous microbial biomass is delipidated by solvent extraction with hexane, ethanol, or mixtures of ethanol and water.

18. The composite of claim 14 or 15, wherein the delipidated biomass optionally comprises a press aid.

19. The composite of claim 18, wherein the press aid is soy hulls.

20. The composite of claim 18 or 19, wherein the press aid is present in an amount of up to 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% by weight of the biomass.

21. The composite of any one of the preceding claims, wherein the biomass optionally containing press aid is present in an amount of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15%, or 20% by weight of the composite.

22. The composite of any one of the preceding claims, wherein the biomass optionally containing press aid has a particle size of less than 3500 microns.

23. The composite of claim 22, wherein the biomass optionally containing press aid has an average particle size of from 0.1 to 1000 microns, or about 500 to 250 microns.

24. The composite of any one of the preceding claims, wherein the biomass optionally containing press aid is pyrolyzed.

25. The composite of any one of the preceding claims, wherein the biomass optionally containing press aid is torrefied.

26. The composite of any one of the preceding claims, wherein the biomass comprises an oleaginous bacteria, oleaginous yeast, or oleaginous microalgae.

27. The composite of claim 26, wherein the biomass comprises a heterotrophic oleaginous microalgae.

28. The composite of claim 27, wherein the microalgae is cultivated with sugar from corn, sorghum, sugar cane, sugar beet, or molasses as a carbon source.

29. The composite of claim 28, wherein the microalgae is cultivated on sucrose.

30. The composite of any one of claims 26 to 29, wherein the microalgae is Parachlorella, Prototheca, Auxenochlorella protothecoides, or Chlorella.

31. The composite of claim 30, wherein the microalgae is Prototheca moriformis.

32. The composite of any one of claims 26 to 31 , comprising oil produced by the microalgae, the oil having a fatty acid profile of at least 60% CI 8: 1; or at least 50% combined total amount of CIO, CI 2, and CI 4; or at least 70% combined total amount of C16:0 and C18: l .

33. The composite of claim 32, wherein oil produced by the microalgae has a fatty acid profile of at least 80-85% CI 8:1.

34. The composite of any one of claims 26 to 33, comprising oil produced by the microalgae, the oil having a fatty acid profile of less than 1% or 0.1% polyunsaturated fatty acids.

35. The composite of any one of the preceding claims, wherein the cellulosic filler is selected from the group consisting of a wood fiber, a wood flour, paper, coconut flour, coffee flour, rice hull, bamboo, and soy hull or combinations thereof.

36. The composite of claim 35, wherein the cellulosic filler is eucalyptus or is an oak, pine, or maple wood fiber, flour, or chip.

37. The composite of any one of the preceding claims, wherein the cellulosic filler is present in an amount of up to 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65%> of the composite.

38. The composite of any one of the preceding claims further comprising a lubricant.

39. The composite of claim 38, wherein the lubricant is a metallic or non-metallic stearate.

40. The composite of claim 39, wherein the lubricant is a zinc stearate.

41. The composite of claim 38, wherein the lubricant is a polyester wax, a paraffin wax, a polypropylene wax, a fatty acid derived diamide, ethylene bis-oleamide, a stearate ester, mixed fatty acid esters or amides, or combinations thereof.

42. The composite of claim 41 wherein the lubricant is ethylene bis-stearamide.

43. The composite of any one of the preceding claims further comprising one or more of a mineral filler, a plasticizer, a UV stabilizer, a colorant, a pesticide, a density modifier, an anti-oxidizing agent, and a foaming agent

44. The composite of claim 43, wherein the pesticide is one or more of an anti- microbial agent, a fungicide, and an insecticide.

45. The composite of claim 43, wherein the mineral filler is talc or mica or a combination thereof.

46. An article comprising the composite of any one of the preceding claims.

47. The article of claim 46 selected from the group consisting of flooring material, outdoor decking, wood paneling, window framing material, interior trim material, railing, fencing, siding, shingles, roofing materials, and an automotive part.

48. The article of claim 46, wherein the composite is encased in a capstock.

49. A method for preparing a wood plastic composite, the method comprising a) blending a thermoplastic resin, a cellulosic filler, an optionally thermochemically treated oleaginous microbial biomass, and a coupling agent or a binder to form a mixture; and

b) extruding, injection molding, hot-pressing, or calendaring said mixture to form the wood plastic composite.

50. A method for preparing a wood composite, the method comprising

a) blending a cellulosic filler, an oleaginous microbial biomass, and a binder to form a mixture; and

b) extruding, injection molding, hot-pressing, or calendaring said mixture to form the wood composite, wherein the wood composite is not a wood plastic composite.

51. The wood plastic composite of any of the preceding claims, comprising a blend of:

a) 30 to 70% by weight of the thermoplastic resin;

b) 30 to 70%) by weight the cellulosic filler and the oleaginous microbial biomass, and c) 0.1 to 5%> of the coupling agent.

52. The wood plastic composite of any of the preceding claims, comprising a blend of:

a) 40 to 60% by weight of the thermoplastic resin;

b) 30 to 60%) by weight of the cellulosic filler and the oleaginous microbial biomass, and

c) 0.1 to 5%> by weight of the coupling agent.

53. The wood plastic composite of any of the preceding claims, comprising a blend of:

a) 30 to 45%o by weight of the thermoplastic resin;

b) 50 to 55%o by weight of the cellulosic filler and the oleaginous microbial biomass, and

c) 0.1 to 5%) by weight of the coupling agent.

54. The wood plastic composite of any of the preceding claims, wherein the oleaginous microbial biomass is present in an amount up of to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%> by weight of the composite.

55. The wood plastic composite of any of the preceding claims, wherein the oleaginous microbial biomass is present in an amount up of to 5 to 10% by weight of the composite.