US 20110275118A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0275118A1 De Crecy (43) Pub. Date: Nov. 10, 2011

(54) METHOD OF PRODUCING FATTY ACIDS (52) U.S. Cl...... 435/42:435/257.1: 435/243; 435/41 FOR BIOFUEL, BIODIESEL, AND OTHER (57) ABSTRACT VALUABLE CHEMICALS The present invention relates to a method of producing fatty acids, by inoculating a mixture of at least one of cellulose, (76) Inventor: Fus De Crecy, Gainesville, FL RNicial S. and iA with a microorganism strain and an algaeE. strain, Ario? and growing said inoculatedand either Strains anaerobic-pho under Suc

(21) Appl. No.: 13/123,662 SW1OS1SE. Weesplitres Sa1S. UC citiesa SU ae)1C-- Eg SR condition, the microorganism strain produces (22) PCT Filed: Oct. 9, 2009 extracellulases that hydrolyze cellulose, hemicellulose and lignin, to produce Sugars, such as glucose, cellobiose, Xylose, mannose, galactose, rhamnose, arabinose or other hemicel (86). PCT No.: PCT/USO9/60199. lulose i. that are metabolized by the algae strain which S371 (c)(1) also metabolizes acetic acid, glucose and hemicellulose from (2), (4) Date: Aug. 1, 2011 pretreatment. Then, either under a Subsequent anaerobic-het s 9. erotrophic condition, the microorganism uses cellulose and produces fermentation products, and the algae Strain uses part Related U.S. Application Data of the released Sugars and exhibits a slower growth rate, or pp under a further anaerobic-phototrophic condition, the micro (60) Provisional application No. 61/104,046, filed on Oct. organism uses cellulose and produces fermentation products 9, 2008. and CO, and the algae strain uses the CO and part of the released Sugars and the at least one fermentation product. Under a further aerobic-heterotrophic condition, the algae Publication Classification strain uses the fermentation products produced by the micro organism strain in a previous anaerobic step to produce one or (51) Int. Cl. more fatty acids, and the microorganism strain continues to CI2P 39/00 (2006.01) produce extracellulases. The microorganism and algae strains CI2N I/00 (2006.01) are evolved for tolerance to furfural. The fatty acids can CI2P I/00 (2006.01) optionally be recovered and used for production of biodiesel CI2N I/2 (2006.01) fuel. Patent Application Publication Nov. 10, 2011 Sheet 1 of 6 US 2011/0275118A1

Bio-Ethanol current art

Biomass Preparation

Chipped feedstock

Pretreatment Heating 160-220 C

Cellulose Hemicellulose Tignin, Furfural, Acetic acid

Detoxification O Separation of inhibitors: furfural, acetic acid & Salts

Enzymatic Hydrolysis Hydrolysis of cellulose in: glucose, cello biose, Xylose & other Sugars. Presence of inhibitors: lignin, glucose & cel Supplied by lobiose Genencor or Novozymes

Solids separation e Separation of lignin & unprocessed biomass

Conversion-Fermentation Inoculation of Strain o Fermentation e Strain metabolizes mainly glucose O Inhibition by ethanol (secondary metabolite) o Presence of lignin. o Production of CO

Filtration-centrifugation Waste biomass Distillation / dehydratation s 78°C (172F) Evaporator/scrubber

Patent Application Publication Nov. 10, 2011 Sheet 2 of 6 US 2011/0275118A1

Producing Fatty Acids Upstream Transesterification

Glycerol-1

Culture unit: Using Raw Glycerol as S S W/ Inicroorganism

S S

y Microorganism processing unit: s Filtering Microorganism

Glycerol-1 SS

Chemicals

Reactor unit: Transesterifying Other Applications Fatty Acids y S Separator unit s^

Fig. B O Patent Application Publication Nov. 10, 2011 Sheet 3 of 6 US 2011/0275118A1

Biotork biodiesel pathway 3-A

Chipped Pretreatment feedstock Heating 120-140°C

Cellulose Hemicellulose Lignin, Furfural, Acetic acid

Fermentation-Respiration-Conversion in heterotrophic conditions oInoculation & growth of Strain1 & Strain2 OBoth strains are aerobic AND anaerobic oSymbiosis: successive of aerobic & anaerobic conditions PAerobic: Strain1 produces extracellular cellulase, oAerobic: Strain2 uses acetic acid from pretreat ment + glucose & hemicellulose hydrolyzed by cellulase produced by Strainl Anaerobic: Strain1 use cellulose & produces sec ondary metabolites as acetic acid, ethanol, Succi nate, butirate, pyruvate & other fermentation prod ucts + CO2 Anaerobic: Strain2 uses part of released Sugars oAerobic: Strain2 uses secondary metabolites pro duced by Strain1 in previous anaerobic conditions Strain1 & 2 evolved for tolerance to furfural oStrain1 evolved for tolerance to acetic acid

Cells Recovery Waste oFiltration & drying cells glycerol

Direct Transesterification of dry cells with: OUltraSonication OEthanol or methanol oAcid or base

Patent Application Publication Nov. 10, 2011 Sheet 4 of 6 US 2011/0275118A1

Biotork biodiesel pathway 3-A

Chipped Pretreatment feedstock Heating 120-140°C

Cellulose Hemicellulose Lignin. Furfural, Acetic acid

Fermentation-Respiration-Conversion in heterotrophic conditions oInoculation & growth of Strain1 & Strain2 Both Strains are aerobic AND anaerobic Symbiosis: successive of aerobic & anaerobic conditions OAerobic: Strain1 produces extracellular cellulase, oAerobic: Strain2 uses acetic acid from pretreat ment + glucose & hemicellulose hydrolyzed by cellulase produced by Strain1 oAnaerobic: Strain1 use cellulose & produces sec ondary metabolites as acetic acid, ethanol, Succi nate, butirate, pyruvate & other fermentation prod ucts + CO2 OAnaerobic: Strain2 uses part of released sugars OAerobic: Strain2 uses secondary metabolites pro duced by Strain1 in previous anaerobic conditions oStrain1 & 2 evolved for tolerance to furfural oStrain1 evolved for tolerance to acetic acid

Cells Recovery Waste oFiltration & drying cells glycerol

Direct Transesterification of dry cells with:

Ultrasonication eEthanol or methanol oAcid or base

Patent Application Publication Nov. 10, 2011 Sheet 5 of 6 US 2011/0275118A1

Biotork biodiesel pathway 3-A

Chipped Pretreatment Sulfuric switchgrass Heating 120-200°C Acid 0.5-2%

Cellulose Iemicellulose Lignin. Furfural, Acetic acid

Fermentation-Respiration-Conversion in heterotrophic conditions O EVG41025 a lusarium oxysporum derivative evolved to use pretreated Switchgrass as carbon source in anaerobic environment producing: acetic acid, ethanol, and other fermentation products like Succinate, bu tyrate, pyruvate as byproducts, and evolved for production of more (or more efficient) cellulases in aerobic environment also using pretreated Switchgrass as carbon source. In addition, the strain was evolved for toler ance to furfural & acetic acid and presence of lignin. O EVG17020 a Chlorella protothecoides derivative evolved in heterotrophic environment to use as carbon sources the fermentation products released by EVG41025, acetic acid, ethanol, and other fermentation prod lucts like Succinate, butyrate, pyruvate, any soluble Sugars released by pretreatment of Switchgrass not used by EVG4 102.5, waste glycerol, and any Sugars released by enzymes activity released in aerobic environ ment by EVG4 1025. o Co-inoculation & growth of EVG41025 and EVG17020 in an heterotrophic cnvironment, in the same fer mentOr O Both strains are aerobic AND anaerobic Symbiosis: succession of aerobic & anaerobic conditions, after measurement of acetic acid and for ethanol concentrations in the mcdium O In aerobic conditions: EVG41025 produces extracellular cellulase, Xylanase and EVG 17020 uses acetic acid, ethanol, and other fermentation products like Succinate, butyrate, pyruvate, waste glycerol and any Solu ble sugars released by pretreatment of switchgrass by EVG4102.5, and any sugars released by enzymes activ ity released in aerobic environment by EVG4 1025 In anaerobic conditions: EVG41025 use cellulose & hemicelluloses and produces secondary metabolites as acetic acid, ethanol, succinate, butyrate, pyruvate & other fermentation products + CO, EVG17020 uses part of the released Sugars. O EVG17020 produces 40% and more fatty acid (cell dry weight)

Cells Recovery oDouble Filtration & cell drying

Waste

glycerol Direct Transesterification of dry cells with: Ultrasonication, membrane rupture Ethanol or methanol Acid or base Fig.s 4 Gede)Biodiesel Patent Application Publication Nov. 10, 2011 Sheet 6 of 6 US 2011/0275118A1

Biotork biodiesel pathway 3 Example B

Chipped Pretreatment Sulfuric Switchgrass Heating 120-200°C Acid O.5-2%

Cellulose Hemicellulose Lignin. Furfural, Acetic acid

Fermentation-Respiration-Conversion in heterotrophic conditions O EVG42050 a Fusarium oxysporum derivative evolved to use pretreated Switchgrass as carbon source in an aerobic environment producing: acetic acid, ethanol, and other fermentation products like Succinate, butyrate, pyruvate as byproducts, and evolved for production of more (or more efficient) cellulases in aerobic environ ment also using pretreated Switchgrass as carbon source. In addition, the strain was evolved for tolerance to furfural & acetic acid and presence of lignin, DEVG17075 a Chlorella protothecoides derivative evolved in heterotrophic environment to use as carbon Sources the fermentation products released by EWG42050, acetic acid, ethanol, and other fermentation products like Succinate, butyrate, pyruvate, any Soluble Sugars released by pretreatment of Switchgrass not used by EVG42050, waste glycerol, and any Sugars released by enzymes activity released in aerobic environment by EWG42O5O. oCo-inoculation & growth of EVG42050and EVG17075 in an heterotrophic environment, in the same fermen tOr Both Strains are aerobic AND anaerobic Symbiosis. Succession of aerobic & anaerobic conditions, after measurement of acetic acid and for ethanol concentrations in the medium In aerobic and heterotrophic conditions: EVG42050 produces extracellular cellulase, Xylanase and EVG 17075 uses acetic acid, ethanol, and other fermentation products like succinate, butyrate, pyruvate, waste glycerol and any soluble Sugars released by pretreatment of Switchgrass by EVG42050, and any sugars released by enzymes activity released in aerobic environment by EVG42050. Possible agitation in fermentor. In anaerobic and phototrophic conditions: EVG42050 use cellulose & hemicelluloses and produces secondary metabolites as acetic acid, ethanol, Succinate, butyrate, pyruvate & other fermentation products + CO2, EVG17075 uses part of the released Sugars, and CO as carbon source. No agitation in fermentor e EVG17075 produces 40% and more fatty acid (cell dry weight)

Cells Recovery oDouble Filtration & cell drying Waste glycerol

Direct Transesterification of dry cells with: OUltrasonication, membrane rupture OEthanol Or methanol O Acid or base Fig.& 5 Sodie)Biodiesel US 2011/02751 18 A1 Nov. 10, 2011

METHOD OF PRODUCING FATTY ACDS reaction. However, current hydrolysis processes are expen FOR BIOFUEL, BIODIESEL AND OTHER sive and inefficient. For example, enzymatic hydrolysis pro VALUABLE CHEMICALS cesses require obtaining costly cellulase enzymes from out side Suppliers. BACKGROUND OF THE INVENTION 0011. A further problem in transforming cellulosic prod 0001 Petroleum is a non-renewable resource. As a result, ucts into ethanol is that up to 50% of the available carbon to many people are worried about the eventual depletion of carbon dioxide is inherently lost through the fermentation petroleum reserves in the future. World petroleum resources process. In addition, ethanol is more corrosive than gas and have even been predicted by some to run out by the 21 diesel. As a result, it requires a distinct distribution infrastruc century (Kerr RA, Science 1998, 281, 1128). ture as well as specifically designed engines. Finally, ethanol 0002 This has fostered the expansion of alternative hydro is 20-30% less efficient than fossil gas and as ethanol evapo carbon products such as ethanol or other microbial fermen rates more easily, a higher percentage is lost along the whole tation products from plant derived feed stock and waste. In production and distribution process. fact, current studies estimate that the United States could 0012 A process that could produce biodiesel from cellu easily produce 1 billion dry tons of biomass (biomass feed lose would alleviate the problems associated with ethanol and stock) material (over half of which is waste) per year. This is other biodiesel productions. primarily in the form of cellulosic biomass. 0013 Biodiesel obtained from microorganisms (e.g., 0003 Cellulose is contained in nearly every natural, free algae and bacteria) is also non-toxic, biodegradable and free growing plant, tree, and bush, in meadows, forests, and fields of sulfur. As most of the carbon dioxide released from burning all over the world without agricultural effort or cost needed to biodiesel is recycled from what was absorbed during the make it grow. growth of the microorganisms (e.g., algae and bacteria), it is 0004. It is estimated that these cellulosic materials could believed that the burning of biodiesel releases less carbon be used to produce enough ethanol to replace 30% or more of dioxide than from the burning of petroleum, which releases the US energy needs in 2030. The great advantage of this carbon dioxide from a source that has been previously stored strategy is that cellulose is the most abundant and renewable within the earth for centuries. Thus, utilizing microorganisms carbon Source on earth and its efficient transformation into a for the production of biodiesel may result in lower green useable fuel could solve the world's energy problem. house gases such as carbon dioxide. 0005 Cellulosic ethanol has been researched extensively. 0014 Some species of microorganisms are ideally suited Cellulosic ethanol is chemically identical to ethanol from for biodiesel production due to their high oil content. Certain other sources, such as corn starch or Sugar, but has the advan microorganisms contain lipids and/or other desirable hydro tage that the cellulosic materials are highly abundant and carbon compounds as membrane components, storage prod diverse. However, it differs in that it requires a greater amount ucts, metabolites and sources of energy. The percentages in of processing to make the Sugar monomers available to the which the lipids, hydrocarbon compounds and fatty acids are microorganisms that are typically used to produce ethanol by expressed in the microorganism will vary depending on the fermentation. type of microorganism that is grown. However, some strains 0006 Although cellulose is an abundant plant material have been discovered where up to 90% of their overall mass resource, its rigid structure makes cellulose a difficult starting contain lipids, fatty acids and other desirable hydrocarbon material to process. As a result, an effective pretreatment is compounds (e.g., Botryococcus). needed to liberate the cellulose from the lignin seal and its 00.15 Algae such as Chlorella sp. and Dunaliella are a crystalline structure so as to render it accessible for a Subse source offatty acids for biodiesel that has been recognized for quent hydrolysis step. By far, most pretreatments are done alongtime. Indeed, these eukaryotic microbes produce a high through physical or chemical means. In order to achieve yield of fatty acids (20-80% of dry weight), and can utilize higher efficiency, some researchers seek to incorporate both CO as carbon with a solar energy source. effects. 0016. However, the photosynthetic process is not efficient 0007 To date, the available pretreatment techniques enough to allow this process to become a cost effective include acid hydrolysis, Steam explosion, ammonia fiber biodiesel source. An alternative was to use the organohet expansion, alkaline wet oxidation and oZone pretreatment. erotrophic properties of Algae and have them grow on carbon Besides effective cellulose liberation, an ideal pretreatment Sources Such as glucose. In these conditions, the fatty acid has to minimize the formation of degradation products yield is extremely high and the fatty acids are of a high because of their inhibitory effects on subsequent hydrolysis quality. The rest of the dry weight is mainly constituted of and fermentation processes. proteins. However, the carbon Sources used are too rare and 0008. The presence of inhibitors makes it more difficult to expensive to achieve any commercial viability. produce ethanol. Even though pretreatment by acid hydroly 0017 Lipid and other desirable hydrocarbon compound sis is probably the oldest and most studied pretreatment tech accumulation in microorganisms can occur during periods of nique, it produces several potent inhibitors including furfural environmental stress, including growth under nutrient-defi and hydroxymethylfurfural (HMF) which are by far regarded cient conditions. Accordingly, the lipid and fatty acid con as the most toxic inhibitors present in lignocellulosic tents of microorganisms may vary in accordance with culture hydrolysate. conditions. 0009. The cellulose molecules are composed of long 0018. The naturally occurring lipids and other hydrocar chains of Sugar molecules of various kinds. In the hydrolysis bon compounds in these microorganisms can be isolated process, these chains are broken down to free the Sugar, transesterified to obtain a biodiesel. The transesterification of before it is fermented for alcohol production. a lipid with a monohydric alcohol, in most cases methanol, 0010. There are two major cellulose hydrolysis processes: yields alkyl esters, which are the primary component of i) a chemical reaction using acids, or an ii) an enzymatic biodiesel. US 2011/02751 18 A1 Nov. 10, 2011

0019. The transesterification reaction of a lipid leads to a 0025. The invention relates to symbiotic relationship biodiesel fuel having a similar fatty acid profile as that of the between the microorganism strain and the algae strain during initial lipid that was used (e.g., the lipid may be obtained from growth under alternating environmental conditions: either animal or plant Sources). As the fatty acid profile of the alternating aerobic-heterotrophic and anaerobic-het resulting biodiesel will vary depending on the source of the erotrophic conditions or alternating aerobic-heterotrophic lipid, the type of alkyl esters that are produced from a trans and anaerobic-phototrophic conditions. esterification reaction will also vary. As a result, the proper 0026. The recovered fatty acids can be used to produce ties of the biodiesel may also vary depending on the Source of biofuels, e.g., biodiesel. the lipid. (e.g., see Schuchardt, etal, TRANSESTERIFICA 0027. The invention eliminates the need for costly TION OF VEGETABLE OILS: A REVIEW, J. Braz. Chem. enzymes produced by outside manufacturers that are required Soc., vol. 9, 1, 199-210, 1998 and G. Knothe, FUEL PRO in conventional processes for bio-ethanol production. Also, CESSING TECHNOLOGY, 86, 1059-1070 (2005), each no detoxification step is required in the present invention. incorporated herein by reference). BRIEF DESCRIPTION OF THE DRAWINGS SUMMARY 0020. The present invention relates to a method for pro 0028 FIG. 1. is a flowchart illustrating a conventional ducing fatty acids from biomass, and in particular a method of process for bio-ethanol production. producing fatty acids from biomass and for producing a bio 0029 FIG. 2. is a flowchart illustrating the general process fuel from said fatty acids. In particular, the present invention for fatty acid production, alcohol production, and biofuel relates to a method of producing fatty acids, by inoculating a production according to an embodiment of the invention. biomass mixture of at least one of cellulose, hemicellulose, 0030 FIG. 3. is a flowchart illustrating a specific process and lignin with a microorganism strain and an algae strain, for fatty acid production, alcohol production, and biofuel that are both aerobic and anaerobic, and then growing said production according to an embodiment of the invention, inoculated Strains under heterotrophic condition and along further depicting how the process eliminates the need for Successive aerobic and anaerobic conditions, or growing said detoxification, the need for Supplying outside enzymes as inoculated Strains under Successive aerobic-heterotrophic required in the conventional process for bio-ethanol produc and anaerobic-phototrophic conditions, creating symbiosis tion, and depicts how the process of the invention can be used between the strains. to reduce carbon dioxide production. 0021. In the first case, under a first aerobic condition, the 0031 FIG. 4. is a flowchart illustrating a preferred microorganism strain produces extracellulases that can embodiment of a specific process for fatty acid production, hydrolyze cellulose, hemicellulose and lignin, to produce alcohol production, and biofuel production according to a Sugars. Such as glucose, cellobiose, Xylose, mannose, galac preferred embodiment of the invention. tose, rhamnose, arabinose or other hemicellulose Sugars, that 0032 FIG. 5. is a flowchart illustrating a preferred can be metabolized by the algae Strain which also can metabo embodiment of a specific process for fatty acid production, lize acetic acid from pretreatment. Under a Subsequent alcohol production, CO production and biofuel production anaerobic condition, the microorganism strain can use cellu according to a preferred embodiment of the invention. lose and can produce fermentation products, and the algae strain can use part of the released Sugars and may exhibit a DETAILED DESCRIPTION OF THE INVENTION slower growth rate. Under a further aerobic condition, the algae Strain can use the fermentation products produced by 0033 Reference will now be made in detail to embodi the microorganism strain in the previous anaerobic step and ments of the invention. Examples of embodiments are illus the algae can produce one or more fatty acids that can then be trated in the accompanying drawings. While the invention recovered, and the microorganism strain continues to produce will be described in conjunction with these embodiments, it extracellulases. will be understood that it is not intended to limit the invention 0022. In the second case, under a first aerobic-het to such embodiments. On the contrary, it is intended to cover erotrophic condition, the microorganism strain produces alternatives, modifications, and equivalents as may be extracellulases that can hydrolyze cellulose, hemicellulose included within the spirit and scope of the invention as and lignin, to produce Sugars, such as glucose, cellobiose, defined by the appended claims. Xylose, mannose, galactose, rhamnose, arabinose or other 0034. In the following description, numerous specific hemicellulose Sugars that can be metabolized by the algae details are set forth in order to provide a thorough understand strain which also can metabolize acetic acid, glucose and ing of the present invention. The present invention may be hemicellulose from a pretreatment. Then, under a Subsequent practiced without some or all of these specific details. In other anaerobic-phototrophic condition, the microorganism can instances, well known process operations have not been use cellulose and can produce fermentation products and described in detail in order not to unnecessarily obscure the CO, and the algae strain can use CO and part of the released present invention. Sugars and the at least one fermentation product. Under a 0035. The present invention relates to a method for pro further aerobic-heterotrophic condition, the algae strain can ducing fatty acids for possible use in biofuel production and use the fermentation products produced by the microorgan alcohol production from biomass material. The method ism strain to produce one or more fatty acids, and the micro involves producing fatty acids, by inoculating a biomass mix organism strain continues to produce extracellulases. ture of at least one of cellulose, hemicellulose, and lignin with 0023. In both cases, the microorganism and algae strains a microorganism strain and an algae Strain, that are both are evolved for tolerance to furfural and acetic acid. aerobic and anaerobic, and then growing said inoculated 0024. The microorganism and algae strains are both aero strains under heterotrophic condition and along Successive bic and anaerobic. aerobic and anaerobic conditions, or growing said inoculated US 2011/02751 18 A1 Nov. 10, 2011 strains under Successive aerobic-heterotrophic and anaero and lignin, and thereby produces at least one fermentation bic-phototrophic conditions, creating symbiosis between the product comprising one or more alcohols in whatever het strains. erotrophic or phototrophic condition, and also CO when in 0036. In the first case, under a first aerobic condition, the phototrophic condition, in said mixture, and microorganism strain produces extracellulases that hydrolyze 0047 said at least one algae strain uses CO., part of said at cellulose, hemicellulose and lignin, to produce Sugars. Such least one fermentation product and part of said at least one of as glucose, cellobiose, Xylose, mannose, galactose, rham glucose, cellobiose, Xylose, mannose, galactose, rhamnose, nose, arabinose or other hemicellulose Sugars, that are arabinose or other hemicellulose Sugars produced by said at metabolized by the algae strain which also metabolizes acetic least one microorganism, when in phototrophic environment, acid from pretreatment. Under a Subsequent anaerobic con or said algae strain uses part of said at least one of glucose, dition, the microorganism strain uses cellulose and produces cellobiose, Xylose, mannose, galactose, rhamnose, arabinose fermentation products, and the algae strain uses part of the or other hemicellulose Sugars produced by said at least one released Sugars and exhibits a slower growth rate. Under a microorganism, when in heterotrophic condition; further aerobic condition, the algae strain uses the fermenta 0048 (iv) growing under aerobic and heterotrophic con tion products produced by the microorganism strain in the ditions, wherein: previous anaerobic step and the algae produces one or more 0049 said at least one algae strain metabolizes said at least fatty acids that are then recovered, and the microorganism one fermentation product produced in step (iii) to produce strain continues to produce extracellulases. one or more fatty acids, and 0037. In the second case, under a first aerobic-het 0050 said at least one microorganism continues produc erotrophic condition, the microorganism strain produces ing said one or more cellulases, hemicellulases, and/or lacca extracellulases that hydrolyze cellulose, hemicellulose and ses; and lignin, to produce Sugars, such as glucose, cellobiose, Xylose, 0051 (v) optionally recovering said one or more fatty mannose, galactose, rhamnose, arabinose or other hemicel acids. lulose Sugars that are metabolized by the algae strain which 0052. In one embodiment, the method is performed under also metabolizes acetic acid, glucose and hemicellulose from heterotrophic conditions. In another embodiment, the method a pretreatment. Then, under a Subsequent anaerobic-pho involves further growing under one or more additional Suc totrophic condition, the microorganism uses cellulose and cessive aerobic and anaerobic conditions. produces fermentation products and CO, and the algae strain 0053. In one embodiment, the method of the invention uses CO and part of the released sugars and the at least one does not involve agitation of the mixture during said anaero fermentation product. Under a further aerobic-heterotrophic bic conditions. In another embodiment, the invention there is condition, the algae strain uses the fermentation products optional agitation during said aerobic conditions. In another produced by the microorganism strain to produce one or more embodiment, the method involves further growing under one fatty acids, and the microorganism strain continues to pro or more additional Successive aerobic-heterotrophic and duce extracellulases. anaerobic-phototrophic conditions. 0038. The recovered fatty acids can be used to produce 0054. In a further embodiment, the method method uses biofuels, e.g., biodiesel. all of the CO, so there is no residual CO, released as a 0039. The microorganism and algae strains are pre byproduct of the method of the invention. adapted/evolved to a pretreated medium resulting intolerance 0055. In one embodiment, the microorganism strain is to furfural and acetic acid. evolved for tolerance to furfural and acetic acid, and the algae 0040. More specifically, the invention is directed to a strain is evolved for tolerance to furfural. method of producing fatty acids, by: 0056. The mixture in step (i) can be obtained from biom 0041 (i) inoculating a mixture of at least one of cellulose, ass. Biomass is any organic material made from plants or hemicellulose, and lignin with at least one microorganism animals, including living or recently dead biological material, strain and at least one algae strain, wherein said at least one which can be used as fuel or for industrial production. Most microorganism strain and said at least one algae strain are commonly, biomass refers to plant matter grown for use as aerobic and anaerobic organisms; biofuel, but it also includes plant or animal matter used for 0042 (ii) growing said inoculated strains under aerobic production of fibers, chemicals or heat. Biomass is a renew and heterotrophic conditions, wherein: able energy source. 0.043 said at least one microorganism strain produces one 0057 There are a wide variety of sources of biomass, or more cellulases, hemicellulases and laccases that hydro including tree and grass crops and forestry, agricultural, and lyze at least one of cellulose, hemicellulose and lignin, to urban wastes, all of which can be utilized in the present produce at least one of glucose, cellobiose, Xylose, mannose, invention. Examples of domestic biomass resources include galactose, rhamnose, arabinose or other hemicellulose Sugars agricultural and forestry residues, municipal Solid wastes, in said mixture, and industrial wastes, and terrestrial and aquatic crops. 0044 said at least one algae strain metabolizes acetic acid 0058. There are many types of plants in the world, and produced in a pretreatment step and also metabolizes said at many ways they can be used for energy production. In general least one of glucose, cellobiose, Xylose, mannose, galactose, there are two approaches: growing plants specifically for rhamnose, arabinose or other hemicellulose Sugars produced energy use, and using the residues from plants that are used by said at least one microorganism strain, and; for other things. The type of plant utilized in the present 0045 (iii) growing under anaerobic and either het invention varies from region to region according to climate, erotrophic or phototrophic condition, wherein: Soils, geography, population, and so on. 0046 said at least one microorganism strain continues to 0059 Energy crops (also called “power crops') can be produce one or more cellulases, hemicellulases, and/or lac grown on farms in potentially very large quantities. Trees and cases that hydrolyze at least one of cellulose, hemicellulose, grasses, including those native to a region, are preferred US 2011/02751 18 A1 Nov. 10, 2011 energy crops, but other, less agriculturally Sustainable crops, 0069. Such plant biomass typically comprises: 5-35% lig including corn can also be used. nin; 10-35% hemicellulose; and 10-60% cellulose. 0060 Trees are a good renewable source of biomass for 0070 The plant biomass that can be utilized in the present processing in the present invention. In addition to growing invention include at least one member selected from the very fast, certain trees will grow back after being cut off close group consisting of wood, paper, Straw, leaves, prunings, to the ground (called “coppicing'). This allows trees to be grass, including Switchgrass, miscanthus, hemp, vegetable harvested every three to eight years for 20 or 30 years before pulp, corn, corn Stover, Sugarcane, Sugar beets, Sorghum, replanting. Such trees (also called “short-rotation woody cassava, poplar, willow, potato waste, bagasse, sawdust, and crops”) grow as much as 40 feet high in the years between mixed waste of plant, oil palm (palm oil) and forest mill harvests. In cooler, wetter regions of the northern United Waste. States, varieties of poplar, maple, black locust, and willow are 0071. In one embodiment of the invention, the plant bio preferred. In the warmer Southeast, Sycamore and Sweetgum mass is obtained from at least one plant selected from the are preferred. While in the warmest parts of Florida and group consisting of Switchgrass, corn stover, and mixed California, eucalyptus is likely to grow well. waste of plant. In another embodiment, the plant biomass is 0061 Grasses are a good renewable source of biomass for obtained from Switchgrass, due to its high levels of cellulose. use in the present invention. Thin-Stemmed perennial grasses 0072. It should be noted that any such biomass material are common throughout the United States. Examples include can by utilized in the method of the present invention. Switchgrass, big bluestem, and other native varieties, which 0073. The plant biomass can initially undergo a pretreat grow quickly in many parts of the country, and can be har ment to prepare the mixture utilized in step (i). Pretreatment vested for up to 10 years before replanting. Thick-stemmed is used to alter the biomass macroscopic and microscopic size perennials including Sugar cane and elephant grass can be and structure, as well as Submicroscopic chemical composi grown in hot and wet climates like those of Florida and tion and structure, so hydrolysis of the carbohydrate fraction Hawaii. Annuals. Such as corn and Sorghum, are another type to monomeric Sugars can be achieved more rapidly and with of grass commonly grown for food. greater yields. Common pretreatment procedures are dis 0062 Oil plants are also a good source of biomass for use closed in Nathan Mosier, Charles Wyman, Bruce Dale, Rich in the present invention. Such plants include, for example, ard Elander, Y.Y. Lee, Mark Holtzapple, Michael Ladisch, Soybeans and Sunflowers that produce oil, which can be used “Features of promising technologies for pretreatment of to make biofuels. Another different type of oil crop is microal lignocellulosic biomass. Bioresource Technology. 96, pp. gae. These tiny aquatic plants have the potential to grow 673-686 (2005), herein incorporated by reference, and dis extremely fast in the hot, shallow, saline water found in some cussed below. lakes in the desert Southwest. 0074 Pretreatment methods are either physical or chemi 0063. In this regard, biomass is typically obtained from cal. Some methods incorporate both effects (McMillan, 1994: waste products of the forestry, agricultural and manufacturing Hsu, 1996). For the purposes of classification, steam and industries, which generate plant and animal waste in large water are excluded from being considered chemical agents quantities. for pretreatment since extraneous chemicals are not added to 0064. Forestry wastes are currently a large source of heat the biomass. Physical pretreatment methods include commi and electricity, as lumber, pulp, and paper mills use them to nution (mechanical reduction in biomass particulate size), power their factories. Another large source of wood waste is steam explosion, and hydrothermolysis. Comminution, tree tops and branches normally left behind in the forest after including dry, wet, and vibratory ball milling (Millett et al., timber-harvesting operations. 1979; Rivers and Emert, 1987; Sidiras and Koukios, 1989), and compression milling (Tassinari et al., 1980, 1982) is 0065 Other sources of wood waste include sawdust and Sometimes needed to make material handling easier through bark from sawmills, shavings produced during the manufac Subsequent processing steps. Acids or bases could promote ture of furniture, and organic sludge (or “liquor) from pulp hydrolysis and improve the yield of glucose recovery from and paper mills. cellulose by removing hemicelluloses or lignin during pre 0066. As with the forestry industry, a large volume of crop treatment. Commonly used acid and base include, for residue remains in the field after harvest. Such waste could be example, HSO and NaOH, respectively. Cellulose solvents collected for biofuel production. Animal farms produce many are another type of chemical additive. Solvents that dissolve “wet wastes' in the form of manure. Such waste can be cellulose in bagasse, cornstalks, tall fescue, and orchardgrass collected and used by the present invention to produce fatty resulted in 90% conversion of cellulose to glucose (Ladischet acids for biofuel production. al., 1978; Hamilton et al., 1984) and showed enzyme hydroly 0067. People generate biomass wastes in many forms, sis could be greatly enhanced when the biomass structure is including “urban wood waste' (such as shipping pallets and disrupted before hydrolysis. Alkaline H2O, ozone, organo leftover construction wood), the biodegradable portion of solv (uses Lewis acids, FeCls, (Al)SO in aqueous alcohols), garbage (paper, food, leather, yard waste, etc.) and the gas glycerol, dioxane, phenol, or ethylene glycol are among Sol given off by landfills when waste decomposes. Even our vents known to disrupt cellulose structure and promote sewage can be used as energy; some sewage treatment plants hydrolysis (Wood and Saddler, 1988). Concentrated mineral capture the methane given off by sewage and burn it for heat acids (H50, HCl), ammonia-based solvents (NH, hydra and power, reducing air pollution and emissions of global Zine), aprotic solvents (DMSO), metal complexes (ferric Warming gases. Sodium tartrate, cadoxen, and cuoxan), and wet oxidation also 0068. In one embodiment, the present invention utilizes reduces cellulose crystallinity and disrupt the association of biomass obtained from plants or animals. Such biomass mate lignin with cellulose, as well as dissolve hemicellulose. These rial can be in any form, including for example, chipped feed methods, while effective, are too expensive for now to be stock, plant waste, animal waste, etc. practical when measured against the value of the glucose US 2011/02751 18 A1 Nov. 10, 2011

(approximately 5 g/lb). The following pretreatment methods produces at least one fermentation product comprising one or of steam explosion, liquid hot water, dilute acid, lime, and more alcohols. Also, under the anaerobic and heterotrophic ammonia pretreatments (AFEX), could have potential as conditions, the algae strain uses part of the Sugars, i.e., glu cost-effective pretreatments. cose, cellobiose, Xylose, mannose, galactose, rhamnose, ara 0075. It should be noted that any such pretreatment pro binose or other hemicellulose Sugars produced by said at least cedure can be utilized to alter the biomass to make the mixture one microorganism, thus producing one or more fatty acids. utilized in the invention. In this regard, the microorganism in Otherwise, under anaerobic-phototrophic conditions, the step (i) can be adapted to apply all pretreatment procedures microorganism strain continues to produce cellulases, hemi and their associated residual compound that can include, for cellulases, and/or laccases that hydrolyze one or more of example, furfural, hydroxymethylfurfural (HMF), phenolics cellulose, hemicellulose, and lignin, and thereby produces at like 3,4-dihydroxybenzal-dehyde, 3-methoxy-4-hydroxy least one fermentation product comprising one or more alco benzoic acid, cinnamic acid, anillin, Vanillin alcohol, as well hols and CO in said mixture. Also, under the anaerobic as Sodium combinates like Sodium hydroxide, nitrate combi phototrophic conditions, the at least one algae strain uses part nates or ammonia, depending on the elected pretreatment or all of CO., part or all of said at least one fermentation method. product and part of the Sugars, i.e., glucose, cellobiose, 0076 Acid pretreatment is a common pretreatment proce Xylose, mannose, galactose, rhamnose, arabinose or other dure. Acid pretreatment by acid hydrolysis and heat treatment hemicellulose Sugars produced by said at least one microor can be utilized to produce the mixture inoculated in step (i) of ganism, thus producing one or more fatty acids. the present invention. Any Suitable acid can be used in this I0083. Then, in step (iv), the mixture is grown under a step, so long as the acid hydrolyzes hemicelluloses away from further aerobic and heterotrophic conditions, wherein said at cellulose. Some common acids that can be used include a least one algae strain metabolizes said at least one fermenta mineral acid selected from hydrochloric acid, phosphoric tion product produced in step (iii) to produce one or more acid, sulfuric acid, or sulfurous acid. Sulfuric acid, for fatty acids. Under this additional aerobic-heterotrophic con example, at concentration of about 0.5 to 2.0% is preferred. dition, the at least one microorganism continues producing Suitable organic acids may be carbonic acid, tartaric acid, one or more cellulases, hemicellulases, and/or laccases. citric acid, glucuronic acid, acetic acid, formic acid, or similar I0084. In optional step (v), the one or more fatty acids are mono- or polycarboxylic acids. The acid pretreatment also recovered. typically involves heating the mixture, for example, in a range I0085. Again, in one embodiment, the method is performed of about 70° C. to 500° C., or in a range of about 120° C. to under heterotrophic conditions. 200° C., or in a range of 120° C. to 140° C. I0086 Also, the method comprises growing under one or 0077 Such acid pretreatment procedure can be used to more successive aerobic and anaerobic conditions. generate the mixture utilized in step (i). I0087 Again, in one embodiment, the method of the inven 0078. It should be noted that, when the biomass is obtained tion does not involve agitation of the mixture during said from plants, the mixture comprises at least one of cellulose, anaerobic conditions. In another embodiment, the invention hemicellulose, lignin, furfural and acetic acid. involves optional agitation during said aerobic conditions. In 0079. After the pretreatment procedure, the mixture in another embodiment, the method involves further growing step (i) comprises at least one of cellulose, hemicellulose, and under one or more additional Successive aerobic-het lignin. In step (i), this mixture is inoculated with at least one erotrophic and anaerobic-phototrophic conditions. microorganism Strain and at least one algae strain. 0088. In a further embodiment, the method uses all of the 0080. The strains are grown heterotrophically under alter CO, so there is no residual CO, released as a byproduct of the nating aerobic and anaerobic conditions or under Successive method of the invention. aerobic-heterotrophic and anaerobic-phototrophic condi I0089 Cellulase refers to a group of enzymes which, acting tions. together hydrolyze cellulose, hemicellulose, and/or lignin. It 0081. To start, the strains are first grown under aerobic and is typically referred to as a class of enzymes produced by heterotrophic conditions (step ii). Under aerobic and het microorganisms (i.e., an extracellular cellulase producer), erotrophic conditions, the microorganism Strain produces one Such as archaea, fungi, bacteria, protozoans, that catalyze the or more cellulases, hemicellulases, and/or laccases that cellulolysis (or hydrolysis) of cellulose. However, it should hydrolyze at least one of cellulose, hemicellulose and lignin be noted that there are cellulases produced by other kinds of to produce at least one Sugar, Such as glucose, cellobiose, microorganisms. Xylose, mannose, galactose, rhamnose, arabinose or other 0090. It is important to note that the present invention can hemicellulose Sugars in said mixture. Also, under the aerobic utilize any microorganism strain that is an extracellular and/ and heterotrophic conditions, the at least one algae strain or intracellular cellulase, hemicellulase, and laccase enzyme metabolizes acetic acid, glucose and hemicellulose produced producer microorganism. Such microorganism produces one in a previous pretreatment step and also metabolizes one or or more cellulases selected from the group consisting of more of the glucose, cellobiose, Xylose, mannose, galactose, endoglucanase, exoglucanase, and B-glucosidase, hemicellu rhamnose, arabinose or other hemicellulose Sugars produced lases, and optionally laccase. The extracellular and/or intra by said at least one microorganism strain, and produces fatty cellular cellulase, hemicellulase, and laccase enzyme pro acids. ducer is selected from the group consisting of prokaryote, 0082. Then in step (iii), the mixture is grown under two bacteria, archaea, eukaryote, yeast and fungi. possible anaerobic conditions: either heterotrophically or 0091 Examples of cellulase producing microorganisms phototrophically. Under such anaerobic and heterotrophic that can be utilized in the present invention include those in conditions, the microorganism Strain continues to produce Table 1. cellulases, hemicellulases, and/orlaccases that hydrolyze one 0092. Accordingly, the cellulase enzymes produced by the or more of cellulose, hemicellulose, and lignin, and thereby microorganism can perform enzymatic hydrolysis on the US 2011/02751 18 A1 Nov. 10, 2011 mixture in step (ii). At the end of the enzymatic hydrolysis, produced in the biomass pretreatment step can include, for the resultant medium can contain glucose, cellobiose, acetic example, furfural, 3,4-dihydroxybenzaldehyde, 3-methoxy acid, furfural, lignin, Xylose, arabinose, rhamnose, mannose, 4-hydroxy-benzoic acid, cinnamic acid, Vanillin, Vanillin galactose, and/or other hemicelluloses Sugars. alcohol, acetic acid, lignin and other residual salts or impu 0093. Again, the present invention can utilize any micro rities. organism that is an extracellular and/or intracellular cellulase 0103) In a preferred embodiment, the method of present enzyme producer to produce the requisite cellulase enzymes invention utilizes at least one microorganism that has been for enzymatic hydrolysis in step (ii) and (iv). As such, any evolutionarily modified and specialized for the specific type prokaryote, including bacteria, archaea, and eukaryote, of biomass used. The evolutionarily modified microorganism including fungi, which produces extracellular and/or intrac can metabolize (enzymatic hydrolysis) the pretreated tar ellular cellulase enzymes may be utilized as the microorgan geted biomass more efficiently and Such microorganisms can ism strain. be better able to tolerate residual compounds, for example, 0094. In one embodiment, the extracellular and/or intrac furfural and acetic acid. In this respect, the evolutionarily ellular cellulase producer is a fungus, archaea or bacteria of a modified microorganism has greater tolerance to furfural and genus selected from the group consisting of Humicola, Tri acetic acid as compared to the unmodified wild-type version choderma, Penicillium, Ruminococcus, Bacillus, Cytophaga, of the microorganism. Sporocytophaga, Humicola grisea, Trichoderma harzianum, 0104. The evolutionarily modified microorganism can Trichodermalignorum, Trichoderma reesei, Penicillium ver also produce one or more cellulase and/or laccase enzymes ruculosum, Ruminococcus albus, Bacillus subtilis, Bacillus that are less inhibited by lignin and/or have improved capacity thermoglucosidasius, Cytophaga spp., Sporocytophaga spp., to metabolize lignin. As such, the evolutionarily modified Clostridium lentocellum and Fusarium oxysporum. microorganism can have improved capacity to produce 0095. In addition, a microorganism that is an extracellular enzymes (such as laccase) that metabolize lignin. Thus, the and/or intracellular laccase enzyme producer may also be cellulase, hemicellulase and/or laccase enzymes produced by utilized in the present invention. Accordingly, any prokary the evolutionarily modified microorganism can have greater ote, including bacteria, archaea, and eukaryote, including capacity to metabolize cellulose and hemicelluloses with lig fungi, which produces extracellular and/or intracellular lac nin as compared to the unmodified wild-type version of the case may be utilized as the microorganism strain. In one microorganism. embodiment, the extracellular and/or intracellular laccase 0105. Due to the use of the evolutionarily modified micro producer is a fungus, bacteria or archaea of a genus selected organism, the present invention allows for production of cel from the group consisting of Humicola, Trichoderma, Peni lulases in situ in the mixture/medium. Consequently, there is cillium, Ruminococcus, Bacillus, Cytophaga and Sporocy no need to buy expensive cellulase enzymes from outside tophaga. According to still a further embodiment the extra Suppliers. This reduces operational costs as compared to con cellular and/or intracellular laccase producer can be at least ventional methods for biofuel production. Further, also due to microorganism selected from the group consisting of Humi the use of the evolutionarily modified microorganism, there is colla grisea, Trichoderma harzianum, Trichodermalignorum, no need to wash and detoxify the acid or alkaline pretreated Trichoderma reesei, Penicillium verruculosum, Ruminococ mixture in the present invention to remove furfural, acetic cus albus, Bacillus subtilis, Bacillus thermoglucosidasius, acid, and salts that would normally inhibit biofuel production Cytophaga spp., Sporocytophaga spp., Clostridium lentocel (as in conventional methods). By removing the wash and lum and Fusarium oxysporum. detoxification steps, the present invention can further reduce 0096. Examples of laccase producing microorganisms operational costs as compared to conventional methods for that can be utilized in the present invention include those in biofuel production. Table 2. 0106. It is noted that an evolutionarily modified microor 0097. In one embodiment, the microorganism strain is a ganism is defined as a microorganism that has been modified bacterium, Such as Fusarium oxysporum. by natural selection techniques. These techniques include, for 0098. Again, any microorganism that is an extracellular example, serial transfer, serial dilution, Genetic Engine, con and/or intracellular cellulase enzyme producer or laccase tinuous culture, and chemostat. One method and chemostatic enzyme producer can be utilized in the present to produce the device (the Genetic Engine; which can avoid dilution resis requisite enzymes for the method. Examples include those tance in continuous culture) has been described in U.S. Pat. listed in Tables 1 and 2. No. 6,686, 194-B1, incorporated herein by reference. 0099. In the present invention, the type of microorganism 0107. In one embodiment, the microorganism is evolu can be selected and/or evolved to be specific to the type of tionarily modified by use of the continuous culture procedure plant biomass used. as disclosed in PCT Application No. PCT/US05/05616, or 0100 Such microorganism hydrolyzes cellulose, hemicel U.S. patent application Ser. No. 1 1/508,286, each incorpo lulose, Xylose, mannose, galactose, rhamnose, arabinose or rated herein by reference. other hemicullulose Sugars in the mixture. 0108) By cultivating a microorganism in this manner, ben 0101 Such microorganism metabolizes cellulose and eficial mutations will occur to produce brand new alleles (i.e., thereby produces at least one fermentation product selected variants of genes) that improve an organism's chances of from the group consisting of Acetate, Acetone, 2,3-Butane Survival and/or growth rate in that particular environment. diol, Butanol, Butyrate, CO2, Ethanol, Formate, Glycolate, 0109 AS Such, the microorganism (e.g., fungi, archaea, Lactate, Malate, Propionate, Pyruvate. Succinate, and other algae, or bacteria) of the present invention can constitute a fermentation products. different strain, which can be identified by the mutations 0102 The microorganism strain is tolerant to one or more acquired during the course of culture, and these mutations, compounds produced by the biomass pretreatment proce may allow the new cells to be distinguished from their ances dure. Such as acid or alkaline pretreatment. Such compounds tors genotype characteristics. Thus, one can select new US 2011/02751 18 A1 Nov. 10, 2011 strains of microorganisms by segregating individuals with by the microorganism. And when step (iii) is run in anaerobic improved rates of reproduction through the process of natural phototrophic condition the at least one algae strain can use the selection. released CO and part or all of the fermentation products and 0110 Selection parameters for evolutionarily modifying part of said glucose, cellobiose, Xylose, mannose, galactose, the microorganism. By way of example, the microorganism rhamnose, arabinose or other hemicellulose Sugars produced in step (i) can be evolutionarily modified, through a natural by the microorganism. selection technique, so that through evolution, it evolves to be 0117 Such fermentation products can include Acetate, adapted to use the particular carbon Source selected. This Acetone, 2,3-Butanediol, Butanol, Butyrate, CO2, Ethanol, involves identifying and selecting the fastest growing variant Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, microorganisms, through adaptation in the natural selection Succinate, and Such released Sugars can include glucose, technique utilized (Such as continuous culture), that grow cellobiose, Xylose, mannose, arabinose, rhamnose, galactose faster than wild-type on a particular carbon source. This also and/or other hemicellulose Sugars. includes selecting those variant microorganisms that have 0118. After growing under the anaerobic conditions of improved tolerance to furfural, to acetic acid or to any step (iii), whether heterotrophic or phototrophic, the mixture residual compound when using dilute acid or alkaline pre is grown under further an aerobic-heterotrophic condition in treatment; or selecting variant microorganisms that produce step (iv). Under this additional aerobic-heterotrophic condi one or more cellulase and/or laccase enzymes that are less tion, the algae strain metabolizes the fermentation product inhibited by lignin and/or have improved capacity to metabo produced in step (iii) to produce one or more fatty acids. Also, lize lignin. This would also involve selecting those producing in step (iv), the microorganism strain continues to produce the above-discussed requisite cellulose enzymes. one or more cellulases, hemicellulases, and/or laccases. 0111. It should be noted that, by using such parameters, 0119 Step (v) involves an optional recovery step to any one of the natural selection techniques could be used in recover the fatty acids produced by the algae in step (iv). the present invention to evolutionarily modify the microor I0120 Phototrophic and/or heterotrophic algae can be used ganism in the present invention. in aerobic and/or anerobic environmental conditions. Such 0112 Accordingly, the microorganisms can be evolution algae can use at least one of Acetate, Acetone, 2,3-Butanediol. arily modified in a number of ways so that their growth rate, Butyrate, CO2, Formate, Glycolate, Lactate, Malate, Propi viability, and utility as a biofuel, or other hydrocarbon prod onate, Pyruvate, Succinate, and at least one of glucose, cel uct can be improved. Thus, the microorganisms can be evo lobiose, Xylose, arabinose, rhamnose, galactose, mannose lutionarily modified to enhance their ability to grow on a and other hemicellulose Sugars under conditions so that said particular Substrate, constituted of the biomass and residual algae Strain produces one or more fatty acids. chemical related to chemical pre-treatment if any. In this I0121 The growth of said at least one algae strain is not regard, the microorganisms can be evolutionarily modified inhibited by the presence of one or more of lignin, furfural, for a specific biomass plant and eventually associated residual salts and cellulases enzymes present in the mixture. chemicals. 0.122 The algae strain can also grow in one or more of the 0113. The microorganisms (e.g., fungi, algae or bacteria) conditions selected from the group consisting of aerobic, are preferably naturally occurring and have not been modified anaerobic, phototrophic, and heterotrophic conditions. by recombinant DNA techniques. In other words, it is not I0123. Similar to the microorganism, the algae may be necessary to genetically modify the microorganism to obtain evolutionarily modified (using the natural selection tech a desired trait. Rather, the desired trait can be obtained by niques discussed above) to serve as an improved source of evolutionarily modifying the microorganism using the tech fatty acids, biofuel, biodiesel, and other hydrocarbon prod niques discussed above. Nonetheless, even genetically modi ucts. In this regard, the algae can be cultivated for use as a fied microorganisms can be evolutionarily modified to biofuel, biodiesel, or hydrocarbon based product. increase their growth rate and/or viability by recombinant 0.124 Most algae need some amount of Sunlight, carbon DNA techniques. dioxide, and water. As a result, algae are often cultivated in 0114. In one embodiment of the invention, the microor open ponds and lakes. However, when algae are grown in ganism is anaerobic and aerobic fungus or bacterium, and in Such an “open’ system, the systems are Vulnerable to con particular, Fusarium oxysporum that has been evolutionarily tamination by other algae and bacteria. modified by continuous culture. 0.125. In one embodiment, the present invention can utilize 0115. In the invention, cellulase activity and/or the amount heterotrophic algae (Stanier et al. Microbial World, Fifth of fermentation products can be measured using common Edition, Prentice-Hall, Englewood Cliffs, N.J., 1986, incor techniques, to determine the cellulase activity and quantity of porated herein by reference), which can be grown in a closed the fermentation product in the Supernatant, before proceed reactOr. ing to the next step. 0.126 While a variety of algal species can be used, algae 0116. It should be noted that, in step (iii), i.e., growth that naturally contain a high amount of lipids, for example, under anaerobic conditions, the inoculated microorganism about 15-90%, about 30-80%, about 40-60%, or about strain catalyzes the cellulose into fermentation products (sec 25-60% of lipids by dry weight of the algae is preferred. Prior ondary metabolites). The fermentation products comprise to the work of the present invention, algae that naturally one or more alcohols, also CO when in phototrophic condi contained a high amount of lipids and high amount of bio tion, and soluble Sugars as Xylose, arabinose, rhamnose, man hydrocarbon were associated as having a slow growth rate. nose, galactose, and other hemicelluloses Sugars that can then Evolutionarily modified algae strains can be produced in be used by the algae in step (iv). In step (iii) under anaerobic accordance with the present invention that exhibit an heterotrophic conditions, the at least one algae Strain uses part improved growth rate. of said glucose, cellobiose, Xylose, mannose, galactose, I0127. The conditions for growing the algae can be used to rhamnose, arabinose or other hemicellulose Sugars produced modify the algae. For example, there is considerable evidence US 2011/02751 18 A1 Nov. 10, 2011 that lipid accumulation takes place in algae as a response to microorganism or algae can be one that does not use fermen the exhaustion of the nitrogen Supply in the medium. Studies tation, and as Such much less carbon dioxide is made as a have analyzed samples where nitrogen has been removed by-product in respiration. from the culture medium and observed that while protein 0.134. Also, said at least one algae strain produces no contents decrease under Such conditions, the carbohydrate inhibitory by-product, for growth of said algae. The growth of content increases, which are then followed by an increase in said algae is not inhibited by the presence of one or more of the lipid content of the algae. (Richardson etal, EFFECTS OF lignin, furfural, salts, cellulase enzymes and hemicellulase NITROGEN LIMITATION ON THE GROWTH OF ALGAE enzymes. 0.135 Types of algae that can be utilized in the invention is ON THE GROWTH AND COMPOSITION OF AUNICEL one or more selected from the group consisting of green LULAR ALGAE IN CONTINUOUS CULTURE CONDI algae, red algae, blue-green algae, cyanobacteria and dia TIONS, Applied Microbiology, 1969, volume 18, page 224.5- tOmS. 2250, 1969, incorporated herein by reference). 0.136. It should be noted that the present invention can 0128. The algae can be evolutionarily modified by a num utilize any algae strain that metabolizes said at least one ber of techniques, including, for example, serial transfer, fermentation products, including acetic acid, ethanol, glu serial dilution, genetic engine, continuous culture, and cose, cellobiose, Xylose or other hemicellulose Sugars, pyru chemostat. Any one of these techniques can be used to modify vate and Succinate, under conditions so that said algae Strain the algae. In one embodiment, the algae can be evolutionarily produces one or more fatty acids. modified by continuous culture, as disclosed in PCT Appli 0.137 By way of example, the algae utilized in step (iii) cation No. PCT/US05/05616, or U.S. patent application Ser. can be from the following taxonomic divisions of algae: No. 1 1/508,286, each incorporated herein by reference. 0.138 (1) Division Chlorophyta (green algae); 0129. In doing so, the microorganisms and the algae can 0.139 (2) Division Cyanophyta (blue-green algae); 0140 (3) Division Bacillariophyta (diatoms); be evolutionarily modified in a number of ways so that their 0141 (4) Division Chrysophyta, growth rate, viability, and utility as a biofuel, or other hydro 0.142 (5) Division Xanthophyta, carbon product can be improved. Accordingly, the microor 0.143 (6) Division Cryptophyta, ganisms and algae can be evolutionarily modified to enhance 0144 (7) Division Euglenophyta, their ability to grow on a particular Substrate. (0145 (8) Division Ochrophyta, 0130 Selection parameters for evolutionarily modifying 0146 (9) Division Haptophyta, and the algae. By way of example, the algae in step (iii) can be 0147 (10) Division Dinophyta. evolutionarily modified, through a natural selection tech 0148 More specifically, the algae can be from the follow nique. Such as continuous culture, so that through evolution, ing species of algae, included within the above divisions the algae evolve to be adapted to use the particular carbon (wherein number in parenthesis corresponds to the division): Source selected. This involves identifying and selecting the 0149 Biddulphia (8); fastest growing variant algae, through adaptation in the natu (O150 Pinguiococcus (8); ral selection technique utilized, that grow faster than wild 0151. Skeletonema (8): type on a particular carbon source. This also includes, for 0152 Emiliania (9): example, selecting those algae that use acetic acid as a carbon 0153. Prymnesium (9); Source with improved tolerance to lignin, furfural and salts. It 0154 Crypthecodinium (10); should be noted that, by using Such parameters, any one of the 0155 Anabaenopsis circularis (2): natural selection techniques could be used in the present 0156 Ankistrodesmus braunii (1): invention to evolutionarily modify the algae in the present (O157 A. falcatus (1): invention. 0158 Botrydiopsis intercedens (5): 0131. In the present invention, such evolutionarily modi 0159 Bracteacoccus cinnabarinus (1): fied algae metabolize one or more compounds selected from 0160 B. engadiensis (1): the group consisting of glucose, cellobiose, Xylose, man (0161 B. minor (Chodat) Petrova (1): nose, galactose, rhamnose, arabinose or other hemicellulose (0162 B. terrestris (1): Sugars and/or waste glycerol, and the algae use one or more of 0163 Bracteacoccus sp. (1): the fermentation products as Acetate, Acetone, 2,3-Butane 0164 Bracteacoccus sp. (1): diol, Butanol, Butyrate, CO2, Ethanol, Formate, Glycolate, (0165 Bumilleriopsis brevis (5): Lactate, Malate, Propionate, Pyruvate, Succinate, as a carbon 0166 Chilomonas paramecium (6): Source, under conditions so that said at least one algae strain 0.167 Chlamydobotry's sp. (1): produces one or more fatty acids. Such evolutionarily modi 0168 Chlamydomonas agloeformis (1): fied algae can also grow in one or more of the conditions 0169 C. dysosmos (1): selected from the group consisting of aerobic, anaerobic, 0170 C. mundana Mojave strain Boron strain (1): phototrophic, and heterotrophic conditions. 0171 C. reinhardi (-) strain (1): 0.132. In one embodiment, when the invention is per 0172 Chlorella ellipsoidea (1): formed under aerobic and heterotrophic conditions, the algae 0173 C. protothecoides (1): use respiration. 0.174 C. pyrenoidosa (1): 0133. In step (iv), the algae using the same amount of (0175 C. pyrenoidosa ATCC 7516 (1): carbon Source as an organism producing fermentation by (0176 C. pyrenoidosa C-37-2 (1): product producer, will produce only up to 10% carbon diox 0177 C. pyrenoidosa Emerson (1): ide. In this regard, more Sugar is used by the algae for growth (0178 C. pyrenoidosa 7-11-05(1): than is transformed to carbon dioxide. Alternatively, the (0179 C. vulgaris (1): US 2011/02751 18 A1 Nov. 10, 2011

0180 C. vulgaris ATCC 9765 (1): selected from the group consisting of Monalanthus Salina, 0181 C. vulgaris Emerson (1): Botryococcus Braunii, Chlorella prototecoides, Outirococ 0182 C. vulgaris Pratt-Trealease (1): cus sp., Scenedesmus obliquus, Nannochloris sp.; Dunaliella 0183) C. vulgaris var. viridis (1): bardawil (D. Salina); Navicula pelliculosa, Radiosphaera 0184 Chlorellidium tetrabotrys (5): negevensis, Biddulphia aurita, Chlorella vulgaris, Nitzschia 0185 Chlorocloster engadinensis (5); palea, Ochromonas dannica, Chrorella pyrenoidosa, Peri 0186. Chlorococcum macrostigmatum (1): dinium cinctum, Neochloris Oleabundans, Oocystis polymor 0187. Chlorococcum sp. (1): pha, Chrysochromulina spp.; Scenedesmus acutus, Scene 0188 Chlorogloea fritschii (2): desmus spp.; Chlorella minutissima, Prynnesium parvum, (0189 Chlorogonium elongatum (1): Navicula pelliculosa, Scenedesmus dimorphus, Scotiella sp.: 0190. Coccomyxa elongata (1): Chorella spp.; Euglena gracilis, and Porphyridium cruen (0191) Cyclotella sp. (3): titlin. (0192 Dictyochloris fragrams (1): 0240 Examples of algae that can be utilized in the present 0193 Euglena gracilis (7); invention include those in Tables 3 and 4. 0194 E. gracilis Vischer (7); 0241. In another embodiment, the algae strain is Chlorella 0195 E. gracilis var. bacillaris (7): protothecoides and has been evolutionarily modified by con 0196) E. gracilis var. Saccharophila (7); tinuous culture using the techniques and procedures 0197) Haematococcus pluvialis (1): described above. 0198 Navicula incerta Grun. (3): 0242 Cyanobacteria may also be used with the present 0199. N. pelliculosa (3): invention. Cyanobacteria are prokaryotes (single-celled 0200 Neochloris alveolaris (1): organisms) often referred to as “blue-green algae.” While 0201 N. aquatica Starr (1): most algae is eukaryotic, cyanobacteria is the most common 0202 N. gelatinosa Herndon (1): exception. Cyanobacteria are generally unicellular, but can be 0203 N. pseudoalveolaris Deason (1): found in colonial and filamentous forms. Some of which 0204 Neochloris sp. (1): differentiate into varying roles. For purposes of the claimed 0205 Nitzschia angularis var. affinis (3) (Grun.) perag.: invention, cyanobacteria are considered algae. 0206 N. chlosterium (Ehr.) (3): 0243 Chlorella protothecoides and Dunaliella Salina are 0207 N. curvilineata Hust. (3): species that have been evolutionarily modified, cultivated, 0208 N. filiformis (3): and harvested for production of a biodiesel. 0209 N. frustulum (Kürtz.) (3): 0244. The following publications relate to growing differ 0210 N. laevis Hust. (3): ent types of algae and then harvesting algae for the purpose of 0211 Nostoc muscorum (2): producing biodiesel are incorporated herein by reference: 0212 Ochromonas malhamensis (4): 0245 Xu et al., HIGH QUALITY BIODESEL PRO 0213 Pediastrum boryanum (1): DUCTION FROM A MICROALGA CHLORELLA 0214 P. duplex (1): PROTHECOIDES BY HETEROTROPHIC GROWTH 0215 Polytoma obtusum (1): IN FERMENTERS, Journal of Biotechnology, vol. 126, 0216 P. ocellatum (1): 499-507, 2006, 0217 Puvella (1): 0246 Kessler, Erich, PHYSIOLOGICAL AND BIO 0218 Polytomella caeca (or coeca) (1); CHEMICAL CONTRIBUTIONS TO THE TAX 0219) Prototheca Zopfii (1): ONOMY OF THE GENUS PROTOTHECA, III. UTI 0220 Scenedesmus acuminatus (1): LIZATION OF ORGANIC CARBON AND 0221) S. acutiformis (1); NITROGEN COMPOUNDS, Arch Microbiol, volume 0222 S. costulatus Chod, var. chlorelloides (1): 132, 103-106, 1982, 0223 S. dimorphus (1): 0247 Johnson D, 1987, OVERVIEW OF THE DOE/ 0224 S. obliquus (1); SERI AQUATIC SPECIES PROGRAM FY 1986 0225 S. quadricauda (1): SOLAR ENERGY INSTITUTE, 0226 Spongiochloris excentrica (1): 0248 Pratt et al., PRODUCTION OF PROTEIN AND 0227 S. lamellata Deason (1): LIPID BY CHLORELLA VULGARIS AND CHLO 0228 S. Spongiosus (1): RELLA PYRENOIDOSA, Journal of Pharmaceutical 0229 Spongiochloris sp. (1): Sciences, volume 52, Issue 10,979-984 2006, and 0230 Spongiococcum alabamense (1): 0249 Sorokin, MAXIMUM GROWTH RATES OF 0231 S. excentricum (1): CHLORELLA IN STEADY-STATE AND IN SYN 0232 S. excentricum Deason et Bold (1) CHRONIZED CULTURES, Proc. N.A.S. volume 45, 0233 S. multinucleatum (1): 1740-1743, 1959. 0234 Stichococcus bacillaris (1): (0250 J. E. Zajic and Y. S. Chiu, HETEROTROPHIC 0235 S. subtilis (1): CULTURE OF ALGAE, Biochemical Engineering, 0236 Tolypothrix tenuis (2): Faculty of Engineering Science, University of Western 0237) Tribonema aequale (5); and Ontario, London. 0238 T. minus (5). 0251. By employing the methods of the instant invention, 0239. In one embodiment, the algae can be from Chloro the inoculation and culture of the mixture with the at least one phyta (Chlorella and Prototheca), Prasinophyta (Du algae strain in step (ii) results in the algae metabolizing at maliella), Bacillariophyta (Navicula and Nitzschia), Ochro least one of glucose, cellobiose, Xylose, mannose, galactose, phyta (Ochromonas), Dinophyta (Gyrodinium) and rhamnose, arabinose or other hemicellulose Sugars. In step Euglenozoa (Euglena). More preferably, the algae is one (iii), when in heterotrophic condition the algae strain uses part US 2011/02751 18 A1 Nov. 10, 2011 of the the glucose, cellobiose, Xylose, mannose, galactose, glycerol, microorganisms exhibit little to no growth on the rhamnose, arabinose or other hemicellulose Sugars produced raw glycerol itself However, the microorganism (e.g., algae or the microorganism in step (ii), and when in phototrophic bacteria) can be evolutionarily modified to utilize raw glyc condition the algae strain uses most of the released CO and of erol as a primary carbon Source. the fermentation products and part of the the glucose, cello 0261 The initial test for determining whether a particular biose, Xylose, mannose, galactose, rhamnose, arabinose or type of microorganism will be able to grow in the presence of other hemicellulose Sugars produced the microorganism in raw glycerol is the Refined Glycerol Test. The Refined Glyc step (ii). In step (iv), the algae metabolizes at least one of the erol Test comprises culturing the microorganism in a medium fermentation products, which can include Acetate, Acetone, comprising refined glycerol. The medium utilized in the 2,3-Butanediol, Butanol, Butyrate, CO2, Ethanol, Formate, Refined Glycerol Test may or may not have another carbon Glycolate, Lactate, Malate, Propionate, Pyruvate, Succinate, Source Such as glucose. However, the medium in the Refined underconditions so that said at least one algae strain produces Glycerol Test must contain a sufficient amount of glycerol So one or more compounds, including fatty acids. that it can be determined that the microorganism exhibits a 0252. The present invention involves culturing and grow minimum metabolizing capacity of the microorganism. The ing the evolutionarily modified algae for extracellular and/or medium can contain about 10 ml-50 ml per liter of refined intracellular production of one or more compounds, such as glycerol, about 0.1 ml-100 ml per liter of refined glycerol, or fatty acids, hydrocarbons, proteins, pigments, Sugars, such as about 2 ml-15 ml per liter of refined glycerol. polysaccharides and monosaccharides, and glycerol. 0262. If a positive result (i.e., the microorganism grows in 0253) The resultant fatty acids, hydrocarbons, proteins, the medium) is obtained with the Refined Glycerol Test, the pigments, Sugars, such as polysaccharides and monosaccha microorganism can be evolutionarily modified to grow in a rides, and glycerol in the algae can be used for biofuel, cos medium comprising raw glycerol. The culture medium can metic, alimentary, mechanical grease, pigmentation, and comprise about 10-100% raw glycerol as a carbon source, medical use production. about 20-90% raw glycerol as a carbon source, about 30-75% 0254. In optional step (v), the fatty acids, hydrocarbons, raw glycerol as a carbon source, about 40-75% raw glycerol proteins, pigments, Sugars, such as polysaccharides and as a carbon source, or about 50.01-55% raw glycerol as a monosaccharides, and glycerol are recovered from the algae. carbon Source. Indeed, some strains of microorganisms have The recovery step can be done by conventional techniques been evolutionary modified to grow on a culture medium including one or more of fractionating the algae in the culture containing 100% raw glycerol. to obtain a fraction containing the compound, and other tech 0263. An evolutionarily modified microorganism which niques including filtration-centrifugation, flocculation, Sol produces extracellular and/or intracellular cellulase, hemicel vent extraction, acid and base extraction, ultrasonication, lulase, and laccase obtained in accordance with the present microwave, pressing, distillation, thermal evaporation, invention has a maximum growth rate using the specific car homogenization, hydrocracking (fluid catalytic cracking), bon sources in the pretreated biomass mixture of at least 5%, and drying of said at least one algae strain containing fatty preferably 10%, 15%, 25%, 50%, 75%, 100%, 200%, 25%- acids. 100%, 25%-100%, 50%-150%, 25-200%, more than 200%, 0255. In one embodiment, the resultant supernatant recov more than 300%, or more than 400% greater than microor ered in step (V) can be reused. ganism of the same species that has not been evolutionarily 0256 Moreover, the recovered fatty acids can be option modified to perform in the present invention. ally isolated and chemically treated (e.g., by transesterifica 0264. An evolutionarily modified algae obtained in accor tion), and thereby made into a biofuel (biodiesel) that can be dance with the present invention has a maximum growth rate incorporated into an engine fuel. using, as a carbon Source, the released polysaccharide and 0257. In this regard, the algae strain of the present inven monosaccharide Sugars from step (i) in the pretreated biom tion produces hydrocarbon chains which can be used as feed ass mixture of at least 5%, preferably 10%, 15%, 25%, 50%, stock for hydrocracking in an oil refinery to produce one or 75%, 100%, 200%, 25%-100%, 25%-100%, 50%-150%, more compounds selected from the group consisting of 25-200%, more than 200%, more than 300%, or more than octane, gasoline, petrol, kerosene, diesel and other petroleum 400% greater than algae of the same species that has not been product as solvent, plastic, oil, grease and fibers. evolutionarily modified to perform in the present invention. 0258 Direct transesterification can be performed on cells 0265 While it is envisioned that the most important com of the algae strain to produce fatty acids for biodiesel fuel. mercial use for microorganisms grown from the by-products Methods of direct transesterification are well known and of biodiesel production will be to use the microorganisms include breaking the algae cells, releasing fatty acids and themselves for products such as biofuel, biodiesel, “bio'- transesterification through a base or acid method with metha hydrocarbon products, renewable hydrocarbon products, and nol or ethanol to produce biodiesel fuel. fatty acid based products, the invention is not limited to this 0259 A further advantage of the method of the present embodiment. For example, if the microorganism is an algae, invention is that the algae strain can be adapted to use waste the algae could be grown from the by-products of biofuel glycerol, as a carbon source, produced by the transesterifica production and harvested for use as a food, medicine, and tion reaction without pretreatment or refinement to produce nutritional Supplement. fatty acids for biodiesel production. 0266 The biofuel obtained from the present invention 0260 Raw glycerol is the by-product of a transesterifica may be used directly or as an alternative to petroleum for tion reaction comprising glycerol and impurities Such as fatty certain products. acid components, oily components, acid components, alkali 0267 In another embodiment, the biofuel (e.g., biodiesel) components, Soap components, alcohol component (e.g., of the present invention may be used in a blend with other methanol or ethanol) solvent (N-hexane) salts and/or diols. petroleum products or petroleum alternatives to obtain fuels Due to the number and type of impurities present in raw Such as motor gasoline and distillate fuel oil composition; US 2011/02751 18 A1 Nov. 10, 2011

finished nonfuel products such as solvents and lubricating 0277. In step (i), the mixture was inoculated with an evo oils; and feedstock for the petrochemical industry Such as lutionarily modified microorganism Strain of Fusarium naphtha and various refinery gases. Oxysporum (designated EVG41025) and an evolutionarily 0268 For example, the biofuel as described above may be modified algae Strain of Chlorella protothecoides (designated used directly in, or blended with other petroleum based com EVG17020). The strains were grown under heterotrophic pounds to produce solvents; paints; lacquers; and printing conditions, and under alternating aerobic and anerobic con inks; lubricating oils; grease for automobile engines and other ditions. The conditions and strains are defined below. machinery. Wax used in candy making, packaging, candles, 0278. The modified Fusarium oxysporum strain matches, and polishes; petroleum jelly; asphalt; petroleum (EVG41025) was evolved to metabolize pretreated coke; and petroleum feedstock used as chemical feedstock Switchgrass more efficiently as a carbon Source and pro derived from petroleum principally for the manufacture of duces fermentation products, such as: Acetate, Acetone, chemicals, synthetic rubber, and a variety of plastics. 2,3-Butanediol, Butanol, Butyrate, CO2, Ethanol, For 0269. In a preferred embodiment, biodiesel produced in mate, Glycolate, Lactate, Malate, Propionate, Pyruvate, accordance with the present invention may be used in a diesel Succinate, and other fermentation products. engine, or may be blended with petroleum-based distillate 0279. The modified Fusarium oxysporum strain fuel oil composition at a ratio Such that the resulting petro (EVG41025) was evolved to tolerate furfural and acetic leum substitute may be in an amount of about 5-95%, about acid better and the presense of lignin. The strain pro 15-85%, about 20-80%, about 25-75%, about 35-50%, about duces external cellulase enzymes specific for Switch 50-75%, or about 75-95% by weight of the total composition. graSS. The components may be mixed in any Suitable manner. 0280 Step (ii) involved growth of Fusarium oxysporum 0270. The process of fueling a compression ignition inter (EVG41025) and Chlorella protothecoides nal combustion engine, comprises drawing air into a cylinder (EVG17020) in an aerobic environment. of a compression ignition internal combustion engine; com 0281 Under the aerobic conditions in step (ii), pressing the air by a compression stroke of a piston in the Fusarium oxysporum (EVG41025) produced cellulases, cylinder, injecting into the compressed air, toward the end of hemicellulases and laccases that hydrolyzed cellulose, the compression stroke, a fuel comprising the biodiesel; and hemicellulose and lignin and produced glucose, cello igniting the fuel by heat of compression in the cylinder during biose, Xylose, mannose, galactose, rhamnose, arabinose operation of the compression ignition internal combustion or other hemicellulse sugars that were metabolized by engine. Chlorella protothecoides (EVG17020) that also 0271. In another embodiment, the biodiesel is used as a metabolized acetic acid from the pretreatment. lubricant or in a process of fueling a compression ignition 0282 Step (iii) involved growth under anaerobic con internal combustion engine. ditions. Fusarium oxysporum (EVG41025) produced 0272 Alternatively, the biofuel may be further processed one or more fermentation products and Chlorella pro to obtain other hydrocarbons that are found in petroleum such tothecoides (EVG17020) used part of the sugars pro as paraffins (e.g., methane, ethane, propane, butane, isobu duced by Fusarium oxysporum (EVG41025). tane, pentane, and hexane), aromatics (e.g., benzene and 0283 Step (iv) involved growing under aerobic condi naphthalene), cycloalkanes (e.g., cyclohexane and methyl tions. Chlorella protothecoides (EVG17020) metabo cyclopentane), alkenes (e.g., ethylene, butene, and lized the fermentation products produced in step (iii) to isobutene), alkynes (e.g., acetylene, and butadienes). produce fatty acids, and Fusarium oxysporum 0273. The resulting hydrocarbons can then in turn be used (EVG41025) continues to produce cellulases. in petroleum based products Such as solvents; paints; lac 0284. Chlorella protothecoides (EVG17020) was quers; and printing inks; lubricating oils; grease for automo evolved to heterotrophically use as carbon sources the bile engines and other machinery, wax used in candy making, fermentation products released by EVG41025 and any packaging, candles, matches, and polishes; petroleum jelly; Soluble Sugars released by the enzymatic activity of asphalt; petroleum coke; and petroleum feedstock used as EVG41025. chemical feedstock derived from petroleum principally for 0285 Chlorella Protothecoides (EVG17020) metabo the manufacture of chemicals, synthetic rubber, and a variety lizes: acetic acid, ethanol, and other fermentation prod of plastics. ucts like Succinate, butyrate, pyruvate, waste glycerol, 0274 The following examples are but two embodiments and it uses acetic acid as a carbon source, and any soluble of the invention. It will be apparent that various changes and Sugars released by the pretreatment and fermentation of modifications can be made without departing from the scope Switchgrass. of the invention as defined in the claims. 0286 Presence of lignin, furfural and salts do not inhibit growth. EXAMPLES (0287 Chlorella Protothecoides (EVG17020) produces (0275 One exemplified embodiment of the method of the 40% or more fatty acid (cell dry weight). present invention can be found in the chart in FIG. 4 and is 0288. In the method, the microorganism and the algae discussed below. were grown under heterotrophic conditions and the algae 0276. In this example (A), a plant biomass material of produced fatty acids. chipped Switchgrass was Subjected to pretreatment by acid 0289. In step (v), the algae cells and fatty acids were then hydrolysis (sulfuric acid 0.5 to 2.0%) and heat treatment recovered by filtration and cell drying. (120-200°C.). This pretreatment procedure produced a mix 0290 Direct transesterification was then performed on the ture for use in the above-discussed step (i). This mixture dry cells (ultrasonication, membrane rupture, through a base contained among other things cellulose, hemicellulose, lig or acid method with methanol or ethanol) to produce biodie nin, furfural, and acetic acid. sel fuel. Waste glycerol was also recovered and recycled. The US 2011/02751 18 A1 Nov. 10, 2011 12 resultant biodiesel fuel was then directly used in any diesel ucts like Succinate, butyrate, pyruvate, waste glycerol, engine for cars, trucks, generators, boats, etc. and it uses acetic acid as a carbon source, and any soluble 0291 Another exemplified embodiment of the method of Sugars released by the pretreatment and fermentation of the present invention can be found in the chart in FIG. 5 and Switchgrass. is discussed below. 0302 Presence of lignin, furfural and salts do not inhibit 0292. In this example (B), a plant biomass material of growth. chipped Switchgrass was Subjected to pretreatment by acid 0303 Chlorella Protothecoides (EVG17075) produces hydrolysis (sulfuric acid 0.5 to 2.0%) and heat treatment 40% or more fatty acid (cell dry weight). (120-200°C.). This pretreatment procedure produced a mix 0304. In the method, the microorganism and the algae ture for use in the above-discussed step (i). This mixture were alternatively grown under heterotrophic and pho contained among other things cellulose, hemicellulose, lig totrophic conditions and the algae produced fatty acids. nin, furfural, and acetic acid. 0305. In step (v), the algae cells and fatty acids were then 0293. In step (i), the mixture was inoculated with an evo recovered by filtration and cell drying. lutionarily modified microorganism strain of Fusarium 0306 Direct transesterification was then performed on the Oxysporum (designated EVG42050) and an evolutionarily dry cells (ultrasonication, membrane rupture, through a base modified algae Strain of Chlorella protothecoides (designated or acid method with methanol or ethanol) to produce biodie EVG17075). In steps (ii)-(iv), the strains were grown under sel fuel. Waste glycerol was also recovered and recycled. The aerobic-heterotrophic conditions (step (ii)), and then anaero resultant biodiesel fuel was then directly used in any diesel bic-phototrophic conditions (step (iii)) and then under aero engine for cars, trucks, generators, boats, etc. The method bic-heterotrophic conditions (step (iv)). The conditions and used most of the released CO, so there is little residual CO strains are defined below. released as a byproduct of said method. 0294 The modified Fusarium oxysporum strain 0307 While the invention has been described and pointed (EVG42050) was evolved to metabolize pretreated out in detail with reference to operative embodiments thereof Switchgrass more efficiently as a carbon Source and pro it will be understood by those skilled in the art that various duces fermentation products, such as: Acetate, Acetone, changes, modifications, Substitutions and omissions can be 2,3-Butanediol. Butanol, Butyrate, CO, Ethanol, For made without departing from the spirit of the invention. It is mate, Glycolate, Lactate, Malate, Propionate, Pyruvate, intended, therefore, that the invention embrace those equiva Succinate, and other fermentation products. lents within the scope of the claims which follow. 0295 The modified Fusarium oxysporum strain (EVG42050) was evolved to tolerate furfural and acetic TABLE 1 acid better and the presense of lignin. The strain pro EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, duces external cellulase enzymes specific for Switch ORINTRA-CELLULAR CELLULASE ENZYMES graSS. 0296 Step (ii) involved growth of Fusarium oxysporum Division Organism (EVG42050) and Chlorella protothecoides Archaea Crenarchaeota Caldivirga maquilingensis (EVG17075) in an aerobic-heterotrophic environment. Archaea Crenarchaeota Sulfolobus acidocaidarius 0297 Under aerobic-heterotrophic conditions in step Archaea Crenarchaeota Sulfolobus solfatanicus Archaea Crenarchaeota Thermofilum pendens (ii), Fusarium oxysporum (EVG42050) produced cellu Archaea Euryarchaeota Picrophilus torridus lases, hemicellulases and laccases that hydrolyzed cel Archaea Euryarchaeota Pyrococcus abyssi lulose, hemicellulose and lignin and produced glucose, Archaea Euryarchaeota Pyrococcus furiosus cellobiose, Xylose, mannose, galactose, rhamnose, ara Archaea Euryarchaeota Pyrococcus horikoshi Archaea Euryarchaeota Thermoplasma volcanium binose or other hemicellulse sugars that were then Bacteria Acidobacteria Acidobacterium capsulatin metabolized by Chlorella protothecoides (EVG17075) Bacteria Actinobacteria Acidothermus cellulolyticus that also metabolized acetic acid from the pretreatment. Bacteria Actinobacteria Actinomadura sp. 0298 Step (iii) involved growth under anaerobic-pho Bacteria Actinobacteria Actinomyces sp. Bacteria Actinobacteria Amycolatopsis orientalis totrophic conditions. Fusarium Oxysporum Bacteria Actinobacteria Arthrobacter attrescens (EVG42050) produced one or more fermentation prod Bacteria Actinobacteria Arthrobacter sp. ucts and CO, and Chlorella protothecoides Bacteria Actinobacteria Bifidobacterium adolescentis Bacteria Actinobacteria Bifidobacterium animalis (EVG17075) used most of the CO, metabolized part or Bacteria Actinobacteria Bifidobacterium bifidum all of the fermentation products and used part of the Bacteria Actinobacteria Bifidobacterium longum sugars produced by Fusarium oxysporum (EVG42050). Bacteria Actinobacteria Cellulomonas fimi 0299 Step (iv) involved growing under aerobic-het Bacteria Actinobacteria Cellulomonas fiavigena Bacteria Actinobacteria Cellulomonas pachnodae erotrophic conditions. Chlorella protothecoides Bacteria Actinobacteria Cellulomonastida (EVG17075) metabolized the fermentation products Bacteria Actinobacteria Cellulosimicrobium sp. produced in step (iii) to produce fatty acids, and Bacteria Actinobacteria Clavibacter michiganensis subsp. Fusarium oxysporum (EVG42050) continues to pro michiganensis duce cellulases. Bacteria Actinobacteria Clavibacter michiganensis subsp. Sepedonicus (0300 Chlorella protothecoides (EVG17075) was Bacteria Actinobacteria Frankia aini evolved to heterotrophically use as carbon sources the Bacteria Actinobacteria Frankia sp. fermentation products released by EVG42050 and any Bacteria Actinobacteria Jonesia sp. Bacteria Actinobacteria Kineococci is radio tolerans Soluble Sugars released by the enzymatic activity of Bacteria Actinobacteria Leifsonia xvii subsp. xvii EVG42050. Bacteria Actinobacteria Microbispora bispora (0301 Chlorella Protothecoides (EVG17075) metabo Bacteria Actinobacteria Micromonospora cellulolyticum lizes: acetic acid, ethanol, and other fermentation prod US 2011/02751 18 A1 Nov. 10, 2011

TABLE 1-continued TABLE 1-continued

EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, ORINTRA-CELLULAR CELLULASE ENZYMES ORINTRA-CELLULAR CELLULASE ENZYMES Division Organism Division Organism Bacteria Actinobacteria Mycobacterium abscessus Bacteria Deinococcus- Thermus caidophilus Bacteria Actinobacteria Mycobacterium avium Thermus Bacteria Actinobacteria Mycobacterium avium subsp. Bacteria Dictyoglomi Dictyoglomus thermophilum Paratuberculosis Bacteria Fibrobacteres Fibrobacter intestina is Bacteria Actinobacteria Mycobacterium bovis Bacteria Fibrobacteres Fibrobacter succinogenes Bacteria Actinobacteria Mycobacterium gilvium Bacteria Fibrobacteres Fibrobacter succinogenes subsp. Bacteria Actinobacteria Mycobacterium marinum Sticcinogenes Bacteria Actinobacteria Mycobacterium smegmatis Bacteria Firmicutes Acetivibrio cellulolyticus Bacteria Actinobacteria Mycobacterium sp. Bacteria Firmicutes Alicyclobacilius acidocaidarius Bacteria Actinobacteria Mycobacterium tuberculosis Bacteria Firmicutes Alkaliphilus metailinedigens Bacteria Actinobacteria Mycobacterium ulcerans Bacteria Firmicutes Anoxybacilius kestanbolensis Bacteria Actinobacteria Mycobacterium vanbaalenii Bacteria Firmicutes Bacilius agaradhaerens Bacteria Actinobacteria Mycobacterium vanbaalenii Bacteria Firmicutes Bacilius alcalophilus Bacteria Actinobacteria Nocardioides sp. Bacteria Firmicutes Bacilius amyloiquefaciens Bacteria Actinobacteria Propionibacterium acnes Bacteria Firmicutes Bacilius anthracis Bacteria Actinobacteria Rhodococci is equi Bacteria Firmicutes Bacilius cereus Bacteria Actinobacteria Saccharopolyspora erythraea Bacteria Firmicutes Bacilius circulians Bacteria Actinobacteria Saccharothrix austraiensis Bacteria Firmicutes Bacilius clausii Bacteria Actinobacteria Salinispora arenicola Bacteria Firmicutes Bacilius firmus Bacteria Actinobacteria Salinispora tropica Bacteria Firmicutes Bacilius halodurans Bacteria Actinobacteria Streptomyces ambofaciens Bacteria Firmicutes Bacilius licheniformis Bacteria Actinobacteria Streptomyces avermitiis Bacteria Firmicutes Bacilius piakoriiensis Bacteria Actinobacteria Streptomyces chartreusis Bacteria Firmicutes Bacilius pumilus Bacteria Actinobacteria Streptomyces Chattanoogensis Bacteria Firmicutes Bacilius sp. Bacteria Actinobacteria Streptomyces coelicolor Bacteria Firmicutes Bacilius subtiis Bacteria Actinobacteria Streptomyces fadiae var. Bacteria Firmicutes Bacilius subtilis subsp. subtilis Bacteria Actinobacteria Streptomyces grisetts Bacteria Firmicutes Bacilius thuringiensis serovar alesti Bacteria Actinobacteria Streptomyces griseus Subsp. griseus Bacteria Firmicutes Bacillus thuringiensis serovar canadensis Bacteria Actinobacteria Streptomyces haistedii Bacteria Firmicutes Bacilius thuringiensis serovar Bacteria Actinobacteria Streptomyces lividans darmstadiensis Bacteria Actinobacteria Streptomyces inanchangensis Bacteria Firmicutes Bacilius thuringiensis serovar israelensis Bacteria Actinobacteria Streptomyces olivaceoviridis Bacteria Firmicutes Bacilius thuringiensis serovar morrisoni Bacteria Actinobacteria Streptomyces reticuli Bacteria Firmicutes Bacilius thiringiensis serovarsan diego Bacteria Actinobacteria Streptomyces roseiscleroticits Bacteria Firmicutes Bacilius thiringiensis serovar Sotto Bacteria Actinobacteria Streptomyces sp. Bacteria Firmicutes Bacilius thuringiensis serovar thompsoni Bacteria Actinobacteria Streptomyces thermocyaneoviolace is Bacteria Firmicutes Bacilius thiringiensis serovariochigiensis Bacteria Actinobacteria Streptomyces thermo violacetis Bacteria Firmicutes Butyrivibriofibrisolvens Bacteria Actinobacteria Streptomyces turgidiscabies Bacteria Firmicutes Caldicellulosinuptor saccharolyticus Bacteria Actinobacteria Streptomyces viridosport is Bacteria Firmicutes Caldicellulosinuptorsp. Bacteria Actinobacteria Thermobifida alba Bacteria Firmicutes Clostridium acetobutyllicum Bacteria Actinobacteria Thermobifida fisca Bacteria Firmicutes Clostridium beijerinckii Bacteria Actinobacteria Thermopolyspora fiexiosa Bacteria Firmicutes Clostridium cellulolyticum Bacteria Bacteroidetes Bacteroides ceiliuliosolvens Bacteria Firmicutes Cliostridium cellulovorans Bacteria Bacteroidetes Bacteroides fragilis Bacteria Firmicutes Clostridium difficile Bacteria Bacteroidetes Bacteroides ovatus Bacteria Firmicutes Clostridium io.sui Bacteria Bacteroidetes Bacteroides theiaioiaomicron Bacteria Firmicutes Clostridium longisportin Bacteria Bacteroidetes Bacteroides vulgatus Bacteria Firmicutes Clostridium phytofermenians Bacteria Bacteroidetes Cytophaga hitchinsonii Bacteria Firmicutes Clostridium phytofermenians Bacteria Bacteroidetes Cytophaga xylanolytica Bacteria Firmicutes Clostridium saccharobutyllicum Bacteria Bacteroidetes Flavobacterium johnsoniae Bacteria Firmicutes Clostridium sp. Bacteria Bacteroidetes Flavobacterium psychrophilum Bacteria Firmicutes Cliostridium Stercorarium Bacteria Bacteroidetes Flavobacterium sp. Bacteria Firmicutes Cliostridium thermocelium Bacteria Bacteroidetes Gramella forsetii Bacteria Firmicutes Etibacterium cellulosolvens Bacteria Bacteroidetes Parabacteroides distasonis Bacteria Firmicutes Etibacterium ruminantium Bacteria Bacteroidetes Prevotella bryantii Bacteria Firmicutes Geobacilius caldoxylosilyticus Bacteria Bacteroidetes Prevoteiia ruminicola Bacteria Firmicutes Geobacilius Stearothermophilus Bacteria Bacteroidetes Rhodothermits marinus Bacteria Firmicutes Geobacilius thermodenitrificans Bacteria Chlorobi Chiorobium chiorochromaii Bacteria Firmicutes Geobacilius thermoleovorans Bacteria Chlorobi Pelodictyon luteoium Bacteria Firmicutes Lactobacilius acidophilus Bacteria Chloroflexi Chloroflexus aurantiacus Bacteria Firmicutes Lactobacilius brevis Bacteria Chloroflexi Herpetosiphon attrantiacus Bacteria Firmicutes Lactobacilitisgasseri Bacteria Chloroflexi Roseiflexus castenholzii Bacteria Firmicutes Lactobacilius johnsonii Bacteria Chloroflexi Roseifiext is sp. Bacteria Firmicutes Lactobacilius rentieri Bacteria Cyanobacteria Anabaena variabilis Bacteria Firmicutes Lactococci is lactis Subsp. Cremoris Bacteria Cyanobacteria Nostoc punctiforme Bacteria Firmicutes Lactococci is lactis Subsp. lactis Bacteria Cyanobacteria Nostoc sp. Bacteria Firmicutes Leticonostoc mesenteroides Subsp. Bacteria Cyanobacteria Synechococci is elongatus Mesenteroides Bacteria Cyanobacteria Synechococci is sp. Bacteria Firmicutes Listeria innoctia Bacteria Cyanobacteria Synechocystis sp. Bacteria Firmicutes Listeria monocytogenes Bacteria Deinococcus- Deinococci is geothermalis Bacteria Firmicutes Paenibacilius barcinonensis Thermus Bacteria Firmicutes Paenibacilius curdlanolyticus US 2011/02751 18 A1 Nov. 10, 2011 14

TABLE 1-continued TABLE 1-continued

EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, ORINTRA-CELLULAR CELLULASE ENZYMES ORINTRA-CELLULAR CELLULASE ENZYMES Division Organism Division Organism Bacteria Firmicutes Paenibacilius fukuinensis Bacteria Proteobacteria Enterobacter sp. Bacteria Firmicutes Paenibacilius lattius Bacteria Proteobacteria Erwinia carotovora Bacteria Firmicutes Paenibacilius pabuli Bacteria Proteobacteria Erwinia carotovora Subsp. AtroSeptica Bacteria Firmicutes Paenibacilius polymyxa Bacteria Proteobacteria Erwinia chrysanthemi Bacteria Firmicutes Paenibacilius sp. Bacteria Proteobacteria Erwinia rhapontici Bacteria Firmicutes Ruminococcus aibus Bacteria Proteobacteria Erwinia tasmaniensis Bacteria Firmicutes Raiminococci is fiavefaciens Bacteria Proteobacteria Escherichia coi Bacteria Firmicutes Streptococci is mutans Bacteria Proteobacteria Gluconacetobacter diazotrophicus Bacteria Firmicutes Streptococci is sanguinis Bacteria Proteobacteria Gluconacetobacter xylinus Bacteria Firmicutes Syntrophomonas wolfei Subsp. wolfei Bacteria Proteobacteria Haheila cheiuensis Bacteria Firmicutes Thermoanaerobacter pseudethanolicus Bacteria Proteobacteria Halorhodospira halophila Bacteria Firmicutes Thermoanaerobacter sp. Bacteria Proteobacteria Klebsiella pneumoniae Bacteria Firmicutes Thermoanaerobacter tengcongensis Bacteria Proteobacteria Klebsiella pneumoniae Subsp. pneumoniae Bacteria Firmicutes Thermoanaerobacterium Bacteria Proteobacteria Legionella pneumophia Lens polysaccharolytictim Bacteria Proteobacteria Legionella pneumophia Paris Bacteria Firmicutes Thermoanaerobacterium saccharolyticum Bacteria Proteobacteria Legionella pneumophia str. Corby Bacteria Firmicutes Thermoanaerobacterium sp. Bacteria Proteobacteria Legionella pneumophia Subsp. Bacteria Firmicutes Thermoanaerobacterium Pneumophila thermosulfitrigenes Bacteria Proteobacteria Leptothrix cholodnii Bacteria Firmicutes Thermobacilius xylamilyticus Bacteria Proteobacteria Leptothrix cholodnii Bacteria Fusobacteria Fusobacterium mortiferum Bacteria Proteobacteria Lysobacter sp. Bacteria Planctomycetes Rhodopinellula baitica Bacteria Proteobacteria Maricantis maris Bacteria Proteobacteria Acidiphilium cryptum Bacteria Proteobacteria Marinomonas sp. Bacteria Proteobacteria Acidovorax avenae Subsp. citrilii Bacteria Proteobacteria Mesorhizobium ioti Bacteria Proteobacteria Acinetobacter battmannii Bacteria Proteobacteria Methyliobacilius flagellatus Bacteria Proteobacteria Aeromonas hydrophila Bacteria Proteobacteria Methyliobacterium extorquens Bacteria Proteobacteria Aeromonas hydrophia Subsp. Bacteria Proteobacteria Methyliobacterium radiotolerans hydrophila Bacteria Proteobacteria Methylobacterium sp. Bacteria Proteobacteria Aeromonas punctata Bacteria Proteobacteria Myxococcus xanthus Bacteria Proteobacteria Aeromonas Salmonicida Subsp. Bacteria Proteobacteria Nitrosospira multiformis Saimonicida Bacteria Proteobacteria Parvibaculum ia vamentivorans Bacteria Proteobacteria Agrobacterium timefaciens Bacteria Proteobacteria Pectobacterium carotovorum Bacteria Proteobacteria Alcaligenes sp. Bacteria Proteobacteria Pectobacterium carotovorum atroseptica Bacteria Proteobacteria Anaeromyxobacter dehalogenians Bacteria Proteobacteria Pectobacterium carotovorum subsp. Bacteria Proteobacteria Anaeromyxobacter sp. COOOji Bacteria Proteobacteria Asaia bogorensis Bacteria Proteobacteria Photobacterium profundum Bacteria Proteobacteria Azoarcus sp. Bacteria Proteobacteria Polaromonas sp. Bacteria Proteobacteria Azorhizobium cattlinodans Bacteria Proteobacteria Polynucleobacter sp. Bacteria Proteobacteria Beijerinckia indica subsp. indica Bacteria Proteobacteria Proteus mirabilis Bacteria Proteobacteria Bordeteia avium Bacteria Proteobacteria Pseudoalteromonas atlantica Bacteria Proteobacteria Bradyrhizobium japonicum Bacteria Proteobacteria Pseudoalteromonas atlantica Bacteria Proteobacteria Bruceiia abortus Bacteria Proteobacteria Pseudoalteromonas haloplanktis Bacteria Proteobacteria Bruceiia canis Bacteria Proteobacteria Pseudoalteromonas sp. Bacteria Proteobacteria Bruceiia neitensis Bacteria Proteobacteria Pseudomonas entomophia Bacteria Proteobacteria Bruceiia ovis Bacteria Proteobacteria Pseudomonas fittorescens Bacteria Proteobacteria Bruceiia Suis Bacteria Proteobacteria Pseudomonas puttida Bacteria Proteobacteria Burkholderia ambifaria Bacteria Proteobacteria Pseudomonas sp. Bacteria Proteobacteria Burkholderia ambifaria Bacteria Proteobacteria Pseudomonas Stutzeri Bacteria Proteobacteria Burkholderia cenocepacia Bacteria Proteobacteria Pseudomonas Syringae pv. mori Bacteria Proteobacteria Burkholderia cepacia Bacteria Proteobacteria Pseudomonas Syringae pv. phaseoicola Bacteria Proteobacteria Burkhoideria maiei Bacteria Proteobacteria Pseudomonas Syringae pv. Syringae Bacteria Proteobacteria Burkhoideria multivorans Bacteria Proteobacteria Pseudomonas Syringae pv. Tomato Bacteria Proteobacteria Burkholderia phymatum Bacteria Proteobacteria Psychromonas ingrahamii Bacteria Proteobacteria Burkholderia phytofirmans Bacteria Proteobacteria Ralstonia eutropha Bacteria Proteobacteria Burkholderia pseudomaiei Bacteria Proteobacteria Raistonia metailidurans Bacteria Proteobacteria Burkholderia sp. Bacteria Proteobacteria Ralstonia Solanacearum Bacteria Proteobacteria Burkholderia sp. Bacteria Proteobacteria Ralstonia Syzygii Bacteria Proteobacteria Burkhoideria thailandensis Bacteria Proteobacteria Rhizobium eti Bacteria Proteobacteria Burkhoideria vietnamiensis Bacteria Proteobacteria Rhizobium leguminosarum bv. trifolii Bacteria Proteobacteria Burkhoideria xenovorans Bacteria Proteobacteria Rhizobium sp. Bacteria Proteobacteria Cattiobacter crescenius Bacteria Proteobacteria Rhodobacter sphaeroides Bacteria Proteobacteria Cattiobacter sp. Bacteria Proteobacteria Rhodoferax ferrireducens Bacteria Proteobacteria Cellvibrio japonicus (formerly Bacteria Proteobacteria Rhodopseudomonas palustris Pseudomonas cellulosa) Bacteria Proteobacteria Saccharophagus degradans Bacteria Proteobacteria Ceilvibrio mixtus Bacteria Proteobacteria Salmonella enterica Subsp. arizonae Bacteria Proteobacteria Chronobacterium violiaceum Bacteria Proteobacteria Salmonella typhimurium Bacteria Proteobacteria Citrobacter koseri Bacteria Proteobacteria Serratia proteanachtians Bacteria Proteobacteria Colwellia psychrerythraea Bacteria Proteobacteria Shigella boydii Bacteria Proteobacteria Enterobacter cloacae Bacteria Proteobacteria Shigella flexneri Bacteria Proteobacteria Enterobacter cloacae Bacteria Proteobacteria Shigella sonnei Bacteria Proteobacteria Enterobacter Sakazaki Bacteria Proteobacteria Sinorhizobium medicae US 2011/02751 18 A1 Nov. 10, 2011 15

TABLE 1-continued TABLE 1-continued

EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, ORINTRA-CELLULAR CELLULASE ENZYMES ORINTRA-CELLULAR CELLULASE ENZYMES Division Organism Division Organism Bacteria Proteobacteria Sinorhizobium meioti Eukaryota Ascomycota Chrysosporium lucknowense Bacteria Proteobacteria Sorangitim cellulost in Eukaryota Ascomycota Claviceps purpurea Bacteria Proteobacteria Stigmatella aurantiaca Eukaryota Ascomycota Coccidioides posadasi Bacteria Proteobacteria Teredinibacter turnerae Eukaryota Ascomycota Cochliobolus heterostrophus Bacteria Proteobacteria Thiobacilius denitrificans Eukaryota Ascomycota Coniothyrium minitans Bacteria Proteobacteria Vibrio choieae Eukaryota Ascomycota Corynascus heterothalicus Bacteria Proteobacteria Vibrio fischeri Eukaryota Ascomycota Cryphonectria parasitica Bacteria Proteobacteria Vibrio harveyi Eukaryota Ascomycota Cryptovaisa sp. Bacteria Proteobacteria Vibrio parahaemolyticus Eukaryota Ascomycota Cylindrocarpon sp. Bacteria Proteobacteria Vibrio sp. Eukaryota Ascomycota Daidinia eschscholzii Bacteria Proteobacteria Vibrio vulnificus Eukaryota Ascomycota Debaryomyces hansenii Bacteria Proteobacteria Xanthomonas aibiineans Eukaryota Ascomycota Debaryomyces occidentalis Bacteria Proteobacteria Xanthomonas axonopodispv. Citri Str. Eukaryota Ascomycota Emericeia deseriorum Bacteria Proteobacteria Xanthomonas campestris pv. Campestris Eukaryota Ascomycota Emericeia nidulians Bacteria Proteobacteria Xanthomonas campestris pv. vesicatoria Eukaryota Ascomycota Epichloe festicae Bacteria Proteobacteria Xanthomonas oryzae pv. oryzae Eukaryota Ascomycota Eremothecium gossypii Bacteria Proteobacteria Xylella fastidiosa Eukaryota Ascomycota Fusarium angioides Bacteria Proteobacteria Yersinia enterocolitica subsp. Eukaryota Ascomycota Fusarium chlamydosporum enterocolitica Eukaryota Ascomycota Fusarium culmorum Bacteria Proteobacteria Yersinia enterocolitica subsp. Eukaryota Ascomycota Fusarium equisei enterocolitica Eukaryota Ascomycota Fusarium iaieriiium Bacteria Proteobacteria Yersinia pestis Eukaryota Ascomycota Fusarium oxysportin Bacteria Proteobacteria Yersinia pestis Eukaryota Ascomycota Fusarium poae Bacteria Proteobacteria Yersinia pestis Antiqua Eukaryota Ascomycota Fusarium proliferatum Bacteria Proteobacteria Yersinia pestis biovar Medievalis Eukaryota Ascomycota Fusarium sp. Bacteria Proteobacteria Yersinia pseudotuberculosis Eukaryota Ascomycota Fusarium tricinctum Bacteria Proteobacteria Yersinia pseudotuberculosis Eukaryota Ascomycota Fusarium tidum Bacteria Proteobacteria Zymomonas mobilis subsp. mobilis Eukaryota Ascomycota Fusarium venenaitin Bacteria Spirochaetes Leptospira bifiexa Eukaryota Ascomycota Fusicoccim sp. Bacteria Spirochaetes Leptospira borgpetersenii Eukaryota Ascomycota Geotrichum sp. Bacteria Spirochaetes Leptospira interrogans Eukaryota Ascomycota Gibbereia avenacea Bacteria. Thermotogae Fervidobacterium nodosum Eukaryota Ascomycota Gibberella moniiformis Bacteria. Thermotogae Petrotoga mobilis Eukaryota Ascomycota Gibberella pullicaris Bacteria. Thermotogae Thermotoga lettingae Eukaryota Ascomycota Gibbereia zeae Bacteria. Thermotogae Thermotoga maritima Eukaryota Ascomycota Giociadium catentiatum Bacteria. Thermotogae Thermotoga neapolitana Eukaryota Ascomycota Humicola grisea Bacteria. Thermotogae Thermotoga petrophila Eukaryota Ascomycota Humicola grisea war, thermoidea Bacteria. Thermotogae Thermotoga sp. Eukaryota Ascomycota Humicola insolens Bacteria Verrucomicrobia Opituitus terrae Eukaryota Ascomycota Humicola nigrescens Eukaryota Ascomycota Acremonium cellulolyticus Eukaryota Ascomycota Hypocreatiecorina Eukaryota Ascomycota Acremonium sp. Eukaryota Ascomycota Hypocrea koningii Eukaryota Ascomycota Acremonium thermophilum Eukaryota Ascomycota Hypocrea ixii Eukaryota Ascomycota Alternaria alternata Eukaryota Ascomycota Hypocreapseudokoningii Eukaryota Ascomycota Aspergilius actileatus Eukaryota Ascomycota Hypocreaschweinitzi Eukaryota Ascomycota Aspergiiitisfiavits Eukaryota Ascomycota Hypocrea virens Eukaryota Ascomycota Aspergilius finigatus Eukaryota Ascomycota Kluyveromyces lactis Eukaryota Ascomycota Aspergilius kawachii Eukaryota Ascomycota Lacazia loboi Eukaryota Ascomycota Aspergilius nidians Eukaryota Ascomycota Leptosphaeria machtians Eukaryota Ascomycota Aspergilius niger Eukaryota Ascomycota Macrophonina phaseolina Eukaryota Ascomycota Aspergilius oryzae Eukaryota Ascomycota Magnaporthe grisea Eukaryota Ascomycota Aspergilius Soiae Eukaryota Ascomycota Mailbranchea cinnamonea Eukaryota Ascomycota Aspergilius sp. Eukaryota Ascomycota Melanocarpus Eukaryota Ascomycota Aspergilius Sulphurents Eukaryota Ascomycota Melanocarpus albomyces Eukaryota Ascomycota Aspergilius terretts Eukaryota Ascomycota Nectria haematococca Eukaryota Ascomycota Aspergilius tubingensis Eukaryota Ascomycota Nectria ipomoeae Eukaryota Ascomycota Aspergilius versicolor Eukaryota Ascomycota Neotyphodium ioli Eukaryota Ascomycota Aureobasidium pitilitians var. Eukaryota Ascomycota Neotyphodium sp. melanigentin Eukaryota Ascomycota Neurospora crassa Eukaryota Ascomycota Beltraniella portoricensis Eukaryota Ascomycota Nigrospora sp. Eukaryota Ascomycota Biomectria ochroietica Eukaryota Ascomycota Paecilomyces lilacinus Eukaryota Ascomycota Blumeria graminis Eukaryota Ascomycota Paracoccidioides brasiliensis Eukaryota Ascomycota Botryosphaeria rhodina (various strains) Eukaryota Ascomycota Botryotinia fuckeiana Eukaryota Ascomycota Penicium canescens Eukaryota Ascomycota Candida albicans Eukaryota Ascomycota Penicilium chrysogenium Eukaryota Ascomycota Candida glabrata Eukaryota Ascomycota Penicium citrinum Eukaryota Ascomycota Candida oleophila Eukaryota Ascomycota Penicium decumbens Eukaryota Ascomycota Chaetonidiumpingtungium Eukaryota Ascomycota Penicilium funiculosum Eukaryota Ascomycota Chaetonium brasiliense Eukaryota Ascomycota Penicilium janthinelium Eukaryota Ascomycota Chaetomium thermophilum Eukaryota Ascomycota Penicium occitanis Eukaryota Ascomycota Chaetomium thermophilum var. Eukaryota Ascomycota Penicium oxalicum thermophilum Eukaryota Ascomycota Penicilitim purpurogentin US 2011/02751 18 A1 Nov. 10, 2011 16

TABLE 1-continued TABLE 1-continued

EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, ORINTRA-CELLULAR CELLULASE ENZYMES ORINTRA-CELLULAR CELLULASE ENZYMES Division Organism Division Organism Eukaryota Ascomycota Penicilium simplicissimum Eukaryota Basidiomycota Trametes versicolor Eukaryota Ascomycota Pichia angusta Eukaryota Basidiomycota Ustilago maydis Eukaryota Ascomycota Pichia anomaia Eukaryota Basidiomycota Voivarielia voivacea Eukaryota Ascomycota Pichia guilliermondii Eukaryota Basidiomycota Xylaria hypoxylon Eukaryota Ascomycota Pichia pastoris Eukaryota Chlorophyta Chlorella vulgaris Eukaryota Ascomycota Pichia stipitis Eukaryota Chytridiomycota Anaeromyces sp. Eukaryota Ascomycota Pseudopiectania nigreila Eukaryota Chytridiomycota Neocallimastix frontalis Eukaryota Ascomycota Robiliarda sp. Eukaryota Chytridiomycota Neocallimastix patriciarum Eukaryota Ascomycota Saccharomyces bavants Eukaryota Chytridiomycota Neocallimastix sp. Eukaryota Ascomycota Saccharomyces castelli Eukaryota Chytridiomycota Orpinomyces joyonii Eukaryota Ascomycota Saccharomyces cerevisiae Eukaryota Chytridiomycota Orpinomyces sp. Eukaryota Ascomycota Saccharomyces kluyveri Eukaryota Cnidaria Hydra magnipapillata Eukaryota Ascomycota Saccobolus diluteilus Eukaryota Mycetozoa Dictyostelium discoideum Eukaryota Ascomycota Sarcoscypha Occidentalis Eukaryota Ochrophyta Eisenia andrei Eukaryota Ascomycota Schizosaccharomyces pombe Eukaryota Oomycota Phytophthora cinnamomi Eukaryota Ascomycota Scopulariopsis brevicautiis Eukaryota Oomycota Phytophthora infestans Eukaryota Ascomycota Scytalidium thermophilum Eukaryota Oomycota Phytophthora ramorum Eukaryota Ascomycota Stachybotrys chartarum Eukaryota Oomycota Phytophthora soiae Eukaryota Ascomycota Stachybotrys echinaia Eukaryota Prasinophyta Ostreococcus lucimarinus Eukaryota Ascomycota Staphylotrichum coccosportin Eukaryota Prasinophyta Ostreococcasiatiri Eukaryota Ascomycota Stilbeiia annulata Eukaryota Ascomycota Talaromyces emersonii Eukaryota Zygomycota Mucor circineioides Eukaryota Ascomycota Thermoascus aurantiacus Eukaryota Zygomycota Phycomyces initens Eukaryota Ascomycota Thermoascus aurantiacus var. levisportis Eukaryota Zygomycota Poitrasia circinians Eukaryota Ascomycota Thermomyces ianuginostis Eukaryo a Zygomycota Rhizopus oryzae Eukaryota Ascomycota Thermomyces verrucosus Eukaryota Zygomycota Syncephaiastrum racemosum Eukaryota Ascomycota Thielavia austraiensis Eukaryota Ascomycota Thielavia microspora Eukaryota Ascomycota Thielavia terrestris Eukaryota Ascomycota Trichoderma asperellum TABLE 2 Eukaryota Ascomycota Trichoderma longibrachiatum Eukaryota Ascomycota Trichoderma parceramosum EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, Eukaryota Ascomycota Trichoderma sp. ORINTRA-CELLULAR LACCASE ENZYMES Eukaryota Ascomycota Trichoderma viride Eukaryota Ascomycota Trichophaea Saccata Division Organism Eukaryota Ascomycota Trichothecium roseum Eukaryota Ascomycota Verticilium dahiae Eukaryota Ascomycota Alternaria alternata Eukaryota Ascomycota Verticilium fungicola Eukaryota Ascomycota Artila adeninivorans Eukaryota Ascomycota Verticilium tenerum Eukaryota Ascomycota Ashbya gossypii Eukaryota Ascomycota Voluteia colleiotrichoides Eukaryota Ascomycota Aspergilius finigatus Eukaryota Ascomycota Xylaria polymorpha Eukaryota Ascomycota Aspergilius niger Eukaryota Ascomycota Yarrowia lipolytica Eukaryota Ascomycota Aspergilius oryzae Eukaryota Basidiomycota Agarict is bisports Eukaryota Ascomycota Aspergilius terretts Eukaryota Basidiomycota Armiiliarieia tabescens Eukaryota Ascomycota Botryotinia fuckeiana Eukaryota Basidiomycota Athelia rolfsii Eukaryota Ascomycota Biergeneratia spariinae Eukaryota Basidiomycota Chlorophyllum molybdites Eukaryota Ascomycota Candida albicans Eukaryota Basidiomycota Clitocybe nuda Eukaryota Ascomycota Candida glabrata Eukaryota Basidiomycota Clitopilus printius Eukaryota Ascomycota Chaetomium globosum Eukaryota Basidiomycota Coprinopsis cinerea Eukaryota Ascomycota Chaetomium thermophilum var. Eukaryota Basidiomycota Crinipellis stipitaria thermophilum Eukaryota Basidiomycota Cryptococcusadeiensis Eukaryota Ascomycota Claviceps purpurea Eukaryota Basidiomycota Cryptococcits fiavits Eukaryota Ascomycota Coccidioides inmitis Eukaryota Basidiomycota Cryptococci is neoformans Eukaryota Ascomycota Colleiotrichum lagenarium Eukaryota Basidiomycota Cryptococci is neoformans var. neoformans Eukaryota Ascomycota Corynascus heterothallicus Eukaryota Basidiomycota Cryptococcus sp. Eukaryota Ascomycota Cryphonectria parasitica Eukaryota Basidiomycota Exidia glanditiosa Eukaryota Ascomycota Cryptococciis bacillisportis Eukaryota Basidiomycota Filobasidium floriforme Eukaryota Ascomycota Cryptococcus gaiti (Cryptococci is albidus) Eukaryota Ascomycota Cryptococci is neoformans Eukaryota Basidiomycota Fomitopsis palustris Eukaryota Ascomycota Cryptococci is neoformans var. Eukaryota Basidiomycota Gloeophyllum sepiarium neoformans Eukaryota Basidiomycota Gloeophyllum trabeum Eukaryota Ascomycota Davidieia tassiana Eukaryota Basidiomycota Infundibulicybe gibba Eukaryota Ascomycota Debaryomyces hansenii Eukaryota Basidiomycota Inpex lacients Eukaryota Ascomycota Emericeia nidulians Eukaryota Basidiomycota Leniinitia edodes Eukaryota Ascomycota Fusarium oxysportin Eukaryota Basidiomycota Meripilus giganteus Eukaryota Ascomycota Fusarium oxysportin f sp. lycopersici Eukaryota Basidiomycota Phanerochaete chrysosporium Eukaryota Ascomycota Fusarium proliferatum Eukaryota Basidiomycota Pietroit is saior-Caiut Eukaryota Ascomycota Gaettmannomyces graminis Eukaryota Basidiomycota Pleurotus sp. Eukaryota Ascomycota Gaettmannomyces graminis var. Eukaryota Basidiomycota Polyporus aircularitis graminis Eukaryota Basidiomycota Schizophyllum commune Eukaryota Ascomycota Gaettmannomyces graminis var. tritici Eukaryota Basidiomycota Trametes hirsuta Eukaryota Ascomycota Gibbereia zeae US 2011/02751 18 A1 Nov. 10, 2011 17

TABLE 2-continued TABLE 2-continued

EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, ORINTRA-CELLULAR LACCASE ENZYMES ORINTRA-C ELLULAR LACCASE ENZYMES

Division Organism Division Organism Karyo Ascomyco Glomerella cingulata Karyo Basi iomyco Filobasidiella neoformans Karyo Ascomyco Hortaea acidophila Karyo Basi iomyco Flammulina velutipes Eukaryo Ascomyco Humicoia insolens Eukaryo Basi iomyco Funalia trogii Eukaryo Ascomyco Hypomyces roseius Eukaryo Basi iomyco Ganoderma applanati in Karyo Ascomyco Hypoxylon sp. Karyo Basi iomyco Ganoderma australe Eukaryo Ascomyco Kluyveromyces lactis Eukaryo Basi iomyco Ganoderma formosanum Eukaryo Ascomyco Lachnum spartinae Eukaryo Basi iomyco Ganoderma lucidum Karyo Ascomyco Lactarius biennius Karyo Basi iomyco Ganoderma sp. Eukaryo Ascomyco Lactarius subduticis Eukaryo Basi iomyco Ganoderma tsunodae Eukaryo Ascomyco Melanocarpus albomyces Eukaryo Basi iomyco Gloeophyllum trabeum Eukaryo Ascomyco Morcheiia conica Eukaryo Basi iomyco Grifolia fondosa Karyo Ascomyco Morcheila crassipes Karyo Basi iomyco Gymnoptisfitsipes Eukaryo Ascomyco Morcheia eiata Eukaryo Basi iomyco Gymnopits peronatus Eukaryo Ascomyco Morcheia escuienia Eukaryo Basi iomyco Gyromitra esculenta Eukaryo Ascomyco Morcheila sp. Eukaryo Basi iomyco Halocyphina villosa Karyo Ascomyco Morcheila spongiola Karyo Basi iomyco Hebeioma radicosum Eukaryo Ascomyco Mycosphaerelia sp. Eukaryo Basi iomyco Heterobasidion abietinum Eukaryo Ascomyco Neurospora crassa Eukaryo Basi iomyco Heterobasidion annosum Eukaryo Ascomyco Paracoccidioides brasiiensis Eukaryo Basi iomyco Heterobasidion araucariae Karyo Ascomyco Peniciiium adameizi Karyo Basi iomyco Heterobasidion insulare Eukaryo Ascomyco Penicilium anagasakiense Eukaryo Basi iomyco Heterobasidion parviporum Eukaryo Ascomyco Penicilium expansum Eukaryo Basi iomyco Hypholoma sp. Eukaryo Ascomyco Penicilium simplissimum Eukaryo Basi iomyco Inpex lacients Karyo Ascomyco Peniciiium variabile Karyo Basi iomyco Leniinitia edodes Eukaryo Ascomyco Phaeosphaeria halima Eukaryo Basi iomyco Leniiniis tigrinus Eukaryo Ascomyco Phaeosphaeria spartinicola Eukaryo Basi iomyco Lepista fiaccida Eukaryo Ascomyco Pichia pastoris Eukaryo Basi iomyco Lepistairina Karyo Ascomyco Pleospora spariinae Karyo Basi iomyco Lepista nuda Eukaryo Ascomyco Podospora anserina Eukaryo Basi iomyco Lyophyllum Shimei Karyo Ascomyco Saccharomyces cerevisiae Karyo Basi iomyco Macrolepiota procera Eukaryo Ascomyco Saccharomyces pastoriantis Eukaryo Basi iomyco Macrotyphula iuncea Karyo Ascomyco Schizosaccharomyces pombe Karyo Basi iomyco Malassezia Sympodialis Eukaryo Ascomyco Stagonospora sp. Eukaryo Basi iomyco Marasmius ailiacetis Eukaryo Ascomyco Taiaromyces flavus Eukaryo Basi iomyco Megacolybia platyphylia Karyo Ascomyco Verpa conica Karyo Basi iomyco Mycena cinerella Eukaryo Ascomycota Yarrowia lipolytica Eukaryo Basi iomyco Mycena crocata Eukaryo Basi iomyco Agarict is bisportis Eukaryo Basi iomyco Mycena galopus Eukaryo Basi iomyco Amanita citrina Eukaryo Basi iomyco Mycena rosea Karyo Basi iomyco Amylosteretin areolatin Karyo Basi iomyco Mycena zephirus Eukaryo Basi iomyco Amylostereum chailleti Eukaryo Basi iomyco Pantis rudis Eukaryo Basi iomyco Amylostereum ferreum Eukaryo Basi iomyco Pantis sp. Eukaryo Basi iomyco Amylosteretin laevigatin Eukaryo Basi iomyco Paxiiius involutus Karyo Basi iomyco Amylosterelim sp. Karyo Basi iomyco Peniophora sp. Eukaryo Basi iomyco Athelia rolfsii Eukaryo Basi iomyco Phanerochaete chrysosporium Eukaryo Basi iomyco Attricularia attriculariidae Eukaryo Basi iomyco Phanerochaete flavidoaiba Eukaryo Basi iomyco Auricularia polytricha Eukaryo Basi iomyco Phanerochaete Sordida Karyo Basi iomyco Bierkandera adista Karyo Basi iomyco Phiebia radiata Eukaryo Basi iomyco Bierkandera sp. Eukaryo Basi iomyco Phlebiopsis gigantea Eukaryo Basi iomyco Bondarzewia montana Eukaryo Basi iomyco Piloderma byssinum Eukaryo Basi iomyco Ceriporiopsis rivatiosa Eukaryo Basi iomyco Piriformospora indica Karyo Basi iomyco Ceriporiopsis subvermispora Karyo Basi iomyco Pietrottis Cornucopiae Eukaryo Basi iomyco Cerrena unicolor Eukaryo Basi iomyco Pleurotus eryngii Eukaryo Basi iomyco Climacocystis borealis Eukaryo Basi iomyco Pietrottis Ostreatus Eukaryo Basi iomyco Clitocybe nebularis Eukaryo Basi iomyco Pietroit is pulmonarius Karyo Basi iomyco Clitocybe quercina Karyo Basi iomyco Pietroit is saior-Caiut Eukaryo Basi iomyco Collybia butyriacea Eukaryo Basi iomyco Pietroit is sapidus Eukaryo Basi iomyco Coniophoraptiteana Eukaryo Basi iomyco Pleurotus sp. Florida Eukaryo Basi iomyco Coppinelius Congregatus Eukaryo Basi iomyco Polyporus alveolaris Karyo Basi iomyco Coppinelius disseminatius Karyo Basi iomyco Polyporus ciliatus Eukaryo Basi iomyco Coprinopsis cinerea Eukaryo Basi iomyco Psathyrella corrugis Eukaryo Basi iomyco Coprinopsis cinerea okayama Eukaryo Basi iomyco Psathyrella dicrani Karyo Basi iomyco Corioiopsis gallica Karyo Basi iomyco Psathyrella murcida Eukaryo Basi iomyco Corinaritisfiexipes Eukaryo Basi iomyco Pycnoporus cinnabarinus Eukaryo Basi iomyco Crinipelis sp. Eukaryo Basi iomyco Pycnoport is coccinetis Eukaryo Basi iomyco Cyathus bulleri Eukaryo Basi iomyco Pycnoportis Sanginetis Karyo Basi iomyco Cyathus sp. Karyo Basi iomyco Rigidoportis microportis Eukaryo Basi iomyco Daedalea quercina Eukaryo Basi iomyco Russia atropiirpairea Eukaryo Basi iomyco Dichomitus Squaiens Eukaryo Basi iomyco Russia mairei Eukaryo Basi iomyco Echinodontium japonictim Eukaryo Basi iomyco Russia nigricans Karyo Basi iomyco Echinodontium tinctorium Karyo Basi iomyco Russia ochroietica Eukaryo Basi iomyco Echinodontium tsugicola Eukaryo Basi iomyco Schizophyllum commune US 2011/02751 18 A1 Nov. 10, 2011 18

TABLE 2-continued TABLE 3-continued

EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA-AND, EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA ORINTRA-CELLULAR LACCASE ENZYMES AND, OR INTRA-CELLULAR CELLULASE ENZYMES ALGAE STRAINS Division Organism Division Strain Eukaryota Basidiomycota Spongipelis sp. Eukaryota Basidiomycota Stropharia Squamosa Bacillariophyta Cymbella pusilla Eukaryota Basidiomycota Termitomyces sp. Bacillariophyta Cymbella tumida Eukaryota Basidiomycota Thanatephorus cucumenis Bacillariophyta Denticula kuetzingii Eukaryota Basidiomycota Tameies cervina Bacillariophyta Diadesmis confervacea Eukaryota Basidiomycota Tametes hirsuta Bacillariophyta Diatona tentie var. elongattin Eukaryota Basidiomycota Tametes ochracea Bacillariophyta Dipioneis subovais Eukaryota Basidiomycota Trametes pubescens Baci ariophyta Encyonema minuti in var. pseudogracilis Eukaryota Basidiomycota Trametes sp. Bacillariophyta Entomoneis paludosa Eukaryota Basidiomycota Tametes versicolor Baci arlop hyta Eucocconeis Sp. Bacillariophyta Elinoia curvata Eukaryota Basidiomycota Tametes viliosa Bacillariophyta Elinoia fiexatiosa Eukaryota Basidiomycota Ustilago maydis Bacillariophyta Eunotia formica Eukaryota Basidiomycota Voivarielia voivacea Bacillariacillariophyta Elinoia glacialis Eukaryota Basidiomycota Xeroconus chrysenteron Bacillariophyta Elinoia maior Eukaryota Basidiomycota Xylaria sp. Bacillariophyta Elinoia naegelii Bacillariophyta Elinoia pectinalis Bacillariophyta Elinoia sp. Bacillariophyta Fallacia monoculata TABLE 3 Bacillariophyta Fallacia pygmaea Bacillariophyta Fragiaria capticina EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA- Bacillariophyta Fragilaria crotonensis AND, OR INTRA-CELLULAR CELLULASE ENZYMES Bacillariophyta Fragilariforma virescens ALGAE STRAINS Bacillariophyta Gomphonema affine Bacillariophyta Gomphonema affine var. insigne Division Strain Bacillariophyta Gomphonema anguistatum Bacillariophyta Gomphonema brebissonii Bacillariophyta Achnanthes coarctata Bacillariophyta Gomphonema Carolinense Bacillariophyta Achnanthes inflata Bacillariophyta Gomphonema dichotomum Bacillariophyta Achnanthidium biporomum Bacillariophyta Gomphonema gracile Bacillariophyta Achnanihidium exiguum Bacillariophyta Gomphonema intracatum Bacillariophyta Achnanthidium lanceolatum Bacillariophyta Gomphonema intracatum var. vibrio Bacillariophyta Achnanthidium minutissimum Bacillariophyta Gomphonema parvulum Bacillariophyta Achnanthidium rostratum Bacillariophyta Gomphonema subclavatum var. commutatin Bacillariophyta Amphora coffeaeformis Bacillariophyta Gomphonema subclavatum var. mexicantin Bacillariophyta Amphora coffeiformis Bacillariophyta Gomphonema subtile Bacillariophyta Amphora committata Bacillariophyta Gomphonema truncatum Bacillariophyta Amphora montana Bacillariophyta Gyrosigma actiminati in Bacillariophyta Amphora pedicatius Bacillariophyta Gyrosigma obtusatin Bacillariophyta Amphora Veneta Bacillariophyta Gyrosigma Spenceri var. curvatia Bacillariophyta Anomoeoneis fogedii Bacillariophyta Hantzschia amphioxys Bacillariophyta Anomoeoneis sphaerophora Bacillariophyta Hantzschia amphioxys f. capitata Bacillariophyta Anomoeoneis sphaerophora f. Costata Bacillariophyta Hantzschia amphioxys war. maior Bacillariophyta Asterionelia formosa Bacillariophyta Hantzschia elongata Bacillariophyta Attiacoseira ambigua Bacillariophyta Hantzschia Sigma Bacillariophyta Attiacoseira grantilata Bacillariophyta Hantzschia spectabilis Bacillariophyta Baciliaria paxillifer Bacillariophyta Hantzschia virgata var. gracilis Bacillariophyta Caioneis bacilium Bacillariophyta Lemnicola hungarica Bacillariophyta Caioneisiewisii Bacillariophyta Minutocellis sp. Bacillariophyta Caioneis moiaris Bacillariophyta Navicula abiskoensis Bacillariophyta Caioneis ventricosa Bacillariophyta Navicula anguista Bacillariophyta Campylodiscus clypeus Bacillariophyta Navicula arvensis Bacillariophyta Chaetoceros elimofei Bacillariophyta Navicula capitata Bacillariophyta Chaetoceros gracilis Bacillariophyta Navicula cincta Bacillariophyta Chaetoceros mieieri Bacillariophyta Navicula cryptocephala Bacillariophyta Cocconeis placentitia var. lineata Bacillariophyta Navicula cryptocephala war. Veneta Bacillariophyta Craticulia acconoda Bacillariophyta Navicula decussis Bacillariophyta Craticula cuspidata Bacillariophyta Navicula erifiiga Bacillariophyta Craticula halophila Bacillariophyta Naviculagerlofi Bacillariophyta Ctenophora pulcheila Bacillariophyta Navicula incerta Bacillariophyta Cyclotella choctawatcheeana Bacillariophyta Navicula ibonensis Bacillariophyta Cyclotella meneghiniana Bacillariophyta Navicula meniscuits var. upsaliensis Bacillariophyta Cyclotella quiliensis Bacillariophyta Navicula minima Bacillariophyta Cylindrotheca fusiformis Bacillariophyta Navicula minima var. atomoides Bacillariophyta Cylindrotheca gracilis Bacillariophyta Navicula phyllepia Bacillariophyta Cymatopietira elliptica Bacillariophyta Navicula radiosa Bacillariophyta Cymatopleura librile Bacillariophyta Navicula radiosa f. tenella Bacillariophyta Cymbella aspera Bacillariophyta Navicula radiosa var. tenella Bacillariophyta Cymbella Cistula Bacillariophyta Navicula recens Bacillariophyta Cymbella microcephala Bacillariophyta Navicula reinhardtii Bacillariophyta Cymbella norvegica Bacillariophyta Navicula rhynchocephala var. amphiceros US 2011/02751 18 A1 Nov. 10, 2011 19

TABLE 3-continued TABLE 3-continued

EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA AND, ORINTRA-CELLULAR CELLULASE ENZYMES AND, OR INTRA-CELLULAR CELLULASE ENZYMES ALGAE STRAINS ALGAE STRAINS

Division Strain Division Strain Baci ario ny Naviculia Sainarum Bacillariophyta Stationeis anceps var. gracilis Baci ario ny Naviculia secura Bacillariophyta Stationeis phoenicenteron Baci ario ny Naviculia seminioides Bacillariophyta Stationeis phoenicenteron f, gracilis Baci ario ny Naviculia seminium Bacillariophyta Stauroneis Smithii var. incisa Baci ario ny Navicula subrhynchocephala Bacillariophyta StatiroSira construens Baci ario ny Naviculia tantitia Bacillariophyta Statirosireila pinnata Baci ario ny Naviculia teneioides Bacillariophyta Stenopterobia curvula Baci ario ny Navicula triptinctata Bacillariophyta Stephanodiscus minutiutius Baci ario ny Navicula triptinctata var. Schizomenoides Bacillariophyta Stephanodiscus parvus Baci ario ny Naviculia trivia is Bacillariophyta Siirirelia anguista Baci ario ny Naviculia viridulia war. rosteiata Bacillariophyta Surinella brightweilii Baci ario ny Neidium affine Bacillariophyta Surireia cf. crumena Baci ario ny Neidium affine var. humerus Bacillariophyta Surireia ovais Baci ario ny Neidium affine var. longiceps Bacillariophyta Surireia ovata Baci ario ny Neidium affine var. undulatum Bacillariophyta Siirirelia ovata var. apiculata Baci ario ny Neidium affine var. undulatum Bacillariophyta Siirirella peisonis Baci ario ny Neidium bisulcatum Bacillariophyta Surireia striatuia Baci ario ny Neidium bisulcatum var. subampilatum Bacillariophyta Synedra famelica Baci ario ny Neidium productum Bacillariophyta Synedra radians Baci ario ny Nitzschia acicularis Bacillariophyta Synedra ruimpens Baci ario ny Nitzschia amphibia Bacillariophyta Synedra ulna Baci ario ny Nitzschia amphibioides Bacillariophyta Synedra tina war. Chaseana Baci ario ny Nitzschia communis Bacillariophyta Tabellaria flocculosa Baci ario ny Nitzschia commutata Bacillariophyta ThalassioSira pseudonana Baci ario ny Nitzschia dissipata Bacillariophyta ThalassioSira sp. Baci ario ny Nitzschia gracilis Bacillariophyta Tryblionella apiculata Baci ario ny Nitzschia inearis Bacillariophyta Tryblionella debilis Baci ario ny Nitzschia inearis war. tentiis Bacillariophyta Tryblionella gracilis Baci ario ny Nitzschia nana Bacillariophyta Tryblionella hungarica Baci ario ny Nitzschia ovais Bacillariophyta Tryblionella levidensis Baci ario ny Nitzschia paleacea Cercozoa Chlorarachnion globosum Baci ario ny Nitzschia perminuta Cercozoa Chlorarachnion reptans Baci ario ny Nitzschia reversa hlorophy Acetabularia acetabulum Baci ario ny Nitzschia rosteiata oropny Acetabularia Caliculus Baci ario ny Nitzschia sigma oropny Acetabularia crenitiata Baci ario ny Nitzschia sp. oropny Acetabularia dentata Baci ario ny Nitzschia subtioides oropny Acetabularia fairlowii Baci ario ny Nitzschia terricoia oropny Acetabularia kiineri Baci ario ny Nitzschia vermicularis oropny Acetabularia major Baci ario ny Nitzschia vitrea oropny Acetabularia yukyutensis Baci ario ny Orthoseira dendroieres oropny Acicularia Schenckii Baci ario ny Phaeodactylum tricornutum oropny Actinotaenium habeebense Baci ario ny Pinnularia appendiculata oropny Anadyomene Stellata Baci ario ny Pinnularia biceps oropny Ankistrodesmits angustus Baci ario ny Pinnularia boreais oropny Ankistrodesmits archiatus Baci ario ny Pinnularia brebissonii oropny Ankistrodesmits densus Baci ario ny Pinnularia gibba oropny Ankistrodesmits falcatus var. acicularis Baci ario ny Pinnularia mayeri oropny Ankistrodesmits falcatus var. Stipitatus Baci ario ny Pinnularia mesolepia oropny Ankistrodesmits nannoselene Baci ario ny Pinnularia nodosa oropny Ankistrodesmits pseudobratini Baci ario ny Pinnularia sp. oropny Ankistrodesmits sp. Baci ario ny Pinnularia subcapitata oropny Aphanochaete confervicola Baci ario ny Pinnularia Subcapitata var. Elongata oropny Aphanochaete confervicola var. major Baci ario ny Pinnularia subgibba oropny Aphanochaete elegans Baci ario ny Pinnularia termitina oropny Aphanochaete elegans var. minor Baci ario ny Pinnularia viridiformis oropny Arthrodesmits sp. Baci ario ny Piaconeis clementis oropny Ascochioris multinucleaia Baci ario ny Placoneis eiginensis oropny Asterococci is superbus Baci ario ny Pleurosigma elongatum oropny Astrephonene gubernaculifera Baci ario ny Pleurosira iaevis oropny Atractomorpha echinaia Baci ario ny Pseudostatirosira construens oropny Atractomorpha porcaia Baci ario ny Rhopalodia contoria oropny Axiococci is clingnani Baci ario ny Rhopalodia gibba oropny Axiosphaera vegetata Baci ario ny Scoliopleutra peisonis oropny Basicladia sp. Baci ario ny Seilaphora pupatia oropny Batophora occidentalis Baci ario ny Seilaphora pupatia var. rectangularis oropny Biastophysa rhizopus Baci ario ny Skeletonema Costatum oropny Boergesenia forbesii Baci ario ny Stationeis actita oropny Boodlea composita Baci ario ny Stationeis anceps oropny Boodiea montagnei Baci ario ny Stationeis anceps f, gracilis oropny Borneteila oligospora US 2011/02751 18 A1 Nov. 10, 2011 20

TABLE 3-continued TABLE 3-continued

EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA- EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA AND, ORINTRA-CELLULAR CELLULASE ENZYMES AND, OR INTRA-CELLULAR CELLULASE ENZYMES ALGAE STRAINS ALGAE STRAINS

Division Strain Division Strain Chlorophyta Borneteila sphaerica Chlorophyta Chlamydomonas mutabilis Chlorophyta Borodineliopsis texensis Chlorophyta Chlamydomonas nociigama Chlorophyta Brachiomonas submarina Chlorophyta Chlamydomonas oblonga Chlorophyta Brachiomonas submarina var.putisifera Chlorophyta Chlamydomonas orbicularis Chlorophyta Bracteacocci is aeritis Chlorophyta Chlamydomonas oviformis Chlorophyta Bracteacocci is cohaerans Chlorophyta Chlamydomonas perpusilius Chlorophyta Bracteacocci is giganteus Chlorophyta Chlamydomonas philotes Chlorophyta Bracteacocci is grandis Chlorophyta Chlamydomonas proteus Chlorophyta Bracteacocci is medionucleatus Chlorophyta Chlamydomonas provasolii Chlorophyta Bracteacoccus minor var. deseriorum Chlorophyta Chlamydomonas pseudagioe Chlorophyta Bracteacocci is minor var. glacialis Chlorophyta Chlamydomonas pseudococci in Chlorophyta Bracteacocci is pseudominor Chlorophyta Chlamydomonas pulsatilia Chlorophyta Bulbochaete hioensis Chlorophyta Chlamydomonas pulvinata Chlorophyta Bulbochaete sp. Chlorophyta Chlamydomonas pygmaea Chlorophyta Capsosiphon fitivescens Chlorophyta Chlamydomonas radiata Chlorophyta Carteria crucifera Chlorophyta Chlamydomonas rapa Chlorophyta Carteria eniganetos var. contaminans Chlorophyta Chlamydomonas saiao Chlorophyta Carteria oivieri Chlorophyta Chlamydomonas simplex Chlorophyta Carteria radiosa Chlorophyta Chlamydomonas Smithii Chlorophyta Carteria sp. Chlorophyta Chlamydomonas sp. Chlorophyta Centrosphaera sp. Chlorophyta Chlamydomonas sphaeroides Chlorophyta Cephaleuros parasiticits Chlorophyta Chlamydomonas Stibangulosa Chlorophyta Cephaleuros virescens Chlorophyta Chlamydomonas Stiriseviensis Chlorophyta Chaetomorpha auricoma Chlorophyta Chlamydomonastovei Chlorophyta Chaetomorpha spiralis Chlorophyta Chlamydomonas ulvaensis Chlorophyta Chaetopeitis sp. Chlorophyta Chlamydomonas yellowstonensis Chlorophyta Chaetophora incrassata Chlorophyta Chlamydomonas zebra Chlorophyta Chaetosphaerializim globostin Chlorophyta Chlamydomonas Zimbabwiensis Chlorophyta Chaimasia antiiiana Chlorophyta Chloranomala cup recola Chlorophyta Chamaetrichon capsulatum Chlorophyta Chiorelia anitrata Chlorophyta Characiochioris acuminata Chlorophyta Chiorelia anitrata war. minor Chlorophyta Characiosiphon rivularis Chlorophyta Chiorelia antarctica Chlorophyta Characium acuminatum Chlorophyta Chiorella ap. Chlorophyta Characium bulgariense Chlorophyta Chiorelia autotrophica war. atypica Chlorophyta Characium californicum Chlorophyta Chiorella capsulata Chlorophyta Characium fusiforme Chlorophyta Chiorella fisca var. fisca Chlorophyta Characium hindaki Chlorophyta Chiorella fisca var. vacuolata Chlorophyta Characium oviforme Chlorophyta Chiorella glucotropha Chlorophyta Characium perforatum Chlorophyta Chiorelia initeoviridis Chlorophyta Characium polymorphium Chlorophyta Chiorelia miniata Chlorophyta Characium saccatum Chlorophyta Chiorelia noctuirna Chlorophyta Characium typicum Chlorophyta Chiorella parva Chlorophyta Chlamydomonas aliensworthii Chlorophyta Chiorella regularis var. minima Chlorophyta Chlamydomonas applanata Chlorophyta Chiorella saccharophila Chlorophyta Chlamydomonas asymmetrica Chlorophyta Chiorella saccharophila var. saccharophila Chlorophyta Chlamydomonas callosa Chlorophyta Chiorella sp. Chlorophyta Chlamydomonas Chlamydogama Chlorophyta Chiorella sphaerica Chlorophyta Chlamydomonas cribrium Chlorophyta Chiorella Stigmatophora Chlorophyta Chlamydomonas culieus Chlorophyta Chiorella vulgaris Chlorophyta Chlamydomonas debaryana var. cristata Chlorophyta Chiorella Zofingiensis Chlorophyta Chlamydomonas desmidii Chlorophyta Chlorochytrium lemnae Chlorophyta Chlamydomonas euryale Chlorophyta Chlorocladusatistralasicus Chlorophyta Chlamydomonas eitstigma Chlorophyta Chiorococcaies Chlorophyta Chlamydomonas fimbriata Chlorophyta Chiorococcum acidum Chlorophyta Chlamydomonas gerlofi Chlorophyta Chlorococci in aegyptiactin Chlorophyta Chlamydomonas gigantea Chlorophyta Chlorococci in aquatictim Chlorophyta Chlamydomonas gloeophila var. irregularis Chlorophyta Chiorococcum arenosum Chlorophyta Chlamydomonas gyrus Chlorophyta Chlorococcum citriforme Chlorophyta Chlamydomonas hedleyi Chlorophyta Chiorococcum croceum Chlorophyta Chlamydomonas hydra Chlorophyta Chlorococcum diplobionticum Chlorophyta Chlamydomonas inflexia Chlorophyta Chlorococcum echinozygotum Chlorophyta Chlamydomonas isabeliensis Chlorophyta Chiorococcum eibense Chlorophyta Chlamydomonas leiostraca Chlorophyta Chiorococcuneikhariiense Chlorophyta Chlamydomonas lunata Chlorophyta Chlorococcum gelatinosum Chlorophyta Chlamydomonas melanospora Chlorophyta Chlorococcum granulosum Chlorophyta Chlamydomonas mexicana Chlorophyta Chiorococcum isabeiense Chlorophyta Chlamydomonas minuta Chlorophyta Chiorococcuniacustre Chlorophyta Chlamydomonas minutissima Chlorophyta Chiorococcum ioculatum Chlorophyta Chlamydomonas monadina Chlorophyta Chlorococcum microstigmatum Ch OO nyW8 Chlamydomonas monoica Ch OO nyW8 Chiorococcum nivale US 2011/02751 18 A1 Nov. 10, 2011 21

TABLE 3-continued TABLE 3-continued

EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA- EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA AND, ORINTRA-CELLULAR CELLULASE ENZYMES AND, OR INTRA-CELLULAR CELLULASE ENZYMES ALGAE STRAINS ALGAE STRAINS

Division Strain Division Strain Chlorophyta Chlorococcim novaeangiae Chlorophyta Dictyosphaerium pianctonictim Chlorophyta Chlorococcum oleofaciens Chlorophyta Dipiostation pentagonium Chlorophyta Chlorococcum oviforme Chlorophyta Gonium multicoccum Chlorophyta Chlorococcum paludosum Chlorophyta Gonium Octionarium Chlorophyta Chlorococcum pamirum Chlorophyta Gonium quadratum Chlorophyta Chlorococcum perforatum Chlorophyta Gonium sacculiferum Chlorophyta Chlorococcum perplexum Chlorophyta Gonium sociaie Chlorophyta Chlorococcim pinguidelin Chlorophyta Gonium sociale var. Saccultin Chlorophyta Chlorococcum pulchrum Chlorophyta Gonium sociale var. Sociate Chlorophyta Chlorococcum pyrenoidosum Chlorophyta Gonium viridisiellatum Chlorophyta Chlorococcum refingens Chlorophyta Klebsormidium flaccidum var. cryophila Chlorophyta Chiorococcum reticulatum Chlorophyta Kiebsormidium marinum Chlorophyta Chlorococcum rugosum Chlorophyta Kiebsormidium subtilissimum Chlorophyta Chlorococcim Saisuginet in Chlorophyta Lagerheinia subsalsa Chlorophyta Chlorococcum sphacosum Chlorophyta Mougeotia transeati Chlorophyta Chiorococcum iairense Chlorophyta Murielia aurantiaca Chlorophyta Chiorococcum texanum Chlorophyta Murieia decolor Chlorophyta Chlorococcum typicum Chlorophyta Mychonastes homosphaera Chlorophyta Chlorococcumuliginosum Chlorophyta Natiococcus pyrifornis Chlorophyta Chlorocystis kornmannii Chlorophyta Natiococcits solutta Chlorophyta Chlorocystis westii Chlorophyta Neospongiococcim alabamense Chlorophyta Chlorogonium perforatum Chlorophyta Neospongiococcim butyrostin Chlorophyta Chlorogonium sp. Chlorophyta Neospongiococcim Commatiforme Chlorophyta Chlorogonium tetragamin Chlorophyta Neospongiococcim concentrictim Chlorophyta Chlorogonium tetragamin Chlorophyta Neospongiococcim excentrictim Chlorophyta Chioromonas actinochioris Chlorophyta Neospongiococcim gigantictim Chlorophyta Chioromonas asteroidea Chlorophyta Neospongiococcum irregulare Chlorophyta Chloromonas augustae Chlorophyta Neospongiococcim macropyrenoidosum Chlorophyta Chloromonas brevispina Chlorophyta Neospongiococcim mahieri Chlorophyta Chioromonas carrizoensis Chlorophyta Neospongiococcim mobile Chlorophyta Chloromonas Chenangoensis Chlorophyta Neospongiococcim multinucleatin Chlorophyta Chioromonas clathrata Chlorophyta Neospongiococcim prolifertin Chlorophyta Chlorosarcinopsis Chlorophyta Neospongiococcim punctatin Chlorophyta Chlorosarcinopsis amylophila Chlorophyta Neospongiococcim rugostin Chlorophyta Chlorosarcinopsis arenicola Chlorophyta Neospongiococcim Saccatum Chlorophyta Chlorosarcinopsis auxotrophica Chlorophyta Neospongiococcim Solitarium Chlorophyta Chlorosarcinopsis bastropiensis Chlorophyta Neospongiococcim Sphaerictim Chlorophyta Chlorosarcinopsis deficiens Chlorophyta Neospongiococci in vacuolatin Chlorophyta Chlorosarcinopsis dissociata Chlorophyta Neospongiococcim variabile Chlorophyta Chlorosarcinopsis eremi Chlorophyta Nephrochlamys subsolitaria Chlorophyta Chlorosarcinopsis halophila Chlorophyta Oedogonium anguististontin Chlorophyta Chlorosarcinopsis minor Chlorophyta Oedogonium horisianum Chlorophyta Chlorosarcinopsis negevensis f. ferruguinea Chlorophyta Oedogonium calliandrum Chlorophyta Chlorosarcinopsis negevensis f negevensis Chlorophyta Oedogonium cardiactin Chlorophyta Chlorosarcinopsis pseudominor Chlorophyta Oedogonium donnelli Chlorophyta Chlorosarcinopsis sempervirens Chlorophyta Oedogonium foveolatum Chlorophyta Chlorosarcinopsis sp. Chlorophyta Oedogonizingeniculatin Chlorophyta Chlorosarcinopsis variabilis Chlorophyta Oedogonium sp. Chlorophyta Coeiastrum cambrictim Chlorophyta Oocystis alpina Chlorophyta Coelastrain proboscidenim var. dilatatum Chlorophyta Oocystis apiculata Chlorophyta Coelastrain proboscidenim var. gracile Chlorophyta Oocystis marssonii Chlorophyta Coelastrum sphaericum Chlorophyta Oocystis minutta Chlorophyta Coenochloris pianoconvexia Chlorophyta Oocystis sp Chlorophyta Cosmarium biretum Chlorophyta Pediastrum angulost in Chlorophyta Cosmarium botrytis Chlorophyta Pediastrum boryanum var. cornutum Chlorophyta Cosmaritin connaitin Chlorophyta Pediastrum boryanum var. longicorne Chlorophyta Cosmaritin clictimis Chlorophyta Pediastrum claihratum Chlorophyta Cosmarium debanyi Chlorophyta Pediastrum duplex var. asperim Chlorophyta Cosmarium formosulum Chlorophyta Pediastrum simplex Chlorophyta Cosmaritim impressitium Chlorophyta Pediastrum sp. Chlorophyta Cosmarium margaritiferum Chlorophyta Pithophora sp. Chlorophyta Cosmarium Smolandicum Chlorophyta Pleurastrum erumpens Chlorophyta Cosmarium sp. Chlorophyta Pietrastrum terrestre Chlorophyta Cosmaritim Subcostatin Chlorophyta Pietrastrum terrestre war. indica Chlorophyta Cosmarium subtumidium Chlorophyta Protosiphon botryoides f. parieticola Chlorophyta Cosmarium turpinii Chlorophyta Protosiphon sp. Chlorophyta Crucigenia lauterbornii Chlorophyta Pseudendocionium akinetum Chlorophyta Crucigeniella rectangularis Chlorophyta Pseudendocionium basiiensis Ch OO nyW8 Dictyococcus Schumacherensis Ch OO nyW8 Pseudendoclonium prostratum Ch OO nyW8 Dictyococci is varians Ch OO nyW8 Pseudococcomyxa adhaerens US 2011/02751 18 A1 Nov. 10, 2011 22

TABLE 3-continued TABLE 3-continued

EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA- EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA AND, ORINTRA-CELLULAR CELLULASE ENZYMES AND, OR INTRA-CELLULAR CELLULASE ENZYMES ALGAE STRAINS ALGAE STRAINS

Division Strain Division Strain Chlorophyta Raphidonema Corcontica Cyanobacteria Anabaena aequalis Chlorophyta Raphidonema longiseta Cyanobacteria Anabaena catentia Chlorophyta Raphidonema nivale Cyanobacteria Anabaena cylindrica Chlorophyta Raphidonema sp. Cyanobacteria Anabaenafios-aquae Chlorophyta Raphidonema spiculiforme Cyanobacteria Anabaena inaequalis Chlorophyta Scenedesmus abundans Cyanobacteria Anabaena minutissina Chlorophyta Scenedesmits archiatus Cyanobacteria Anabaena randhawae Chlorophyta Scenedesmits armaints Cyanobacteria Anabaena sp. Chlorophyta Scenedesmits basiliensis Cyanobacteria Anabaena sphaerica Chlorophyta Scenedesmus bijugatus var. Seriatus Cyanobacteria Anabaena spiroides Chlorophyta Scenedesmits breviacitieatus Cyanobacteria Anabaena subcylindrica Chlorophyta Scenedesmus dispar Cyanobacteria Anabaena subtropica Chlorophyta Scenedesmus hystrix Cyanobacteria Anabaena variabilis Chlorophyta Scenedesmulsiovais Cyanobacteria Anabaena verrucosa Chlorophyta Scenedesmits naegelii Cyanobacteria Anacystis marina Chlorophyta Scenedesmits pannonicus Cyanobacteria Aphanizomenon fios-aquae Chlorophyta Scenedesmits parisiensis Cyanobacteria Arthrospira fusiformis Chlorophyta Scenedesmits platydiscus Cyanobacteria Caiothrix anomaia Chlorophyta Scenedesmits sp. Cyanobacteria Caiothrix javanica Chlorophyta Scenedesmits subspicatus Cyanobacteria Caiothrix membranacea Chlorophyta Selena Strain capricornitum Cyanobacteria Caiothrix parietina Chlorophyta Seien.astrum minutum Cyanobacteria Caiothrix sp. Chlorophyta Selena strum sp. Cyanobacteria Chamaesiphon sp. Chlorophyta Sirogonium Sticticum Cyanobacteria Chroococcidiopsis sp. Chlorophyta Spirogyra condensata Cyanobacteria Cylidrospermum sp. Chlorophyta Spirogyra crassispina Cyanobacteria Cylindrospermopsis raciborskii Chlorophyta Spirogyra gracilis Cyanobacteria Cylindrospermum licheniforme Chlorophyta Spirogyra grevilleana Cyanobacteria Cylindrospermum sp. Chlorophyta Spirogyrainiergensii Cyanobacteria Dermocarpa sp. Chlorophyta Spirogyra iiana Cyanobacteria Dermocarpa violacea Chlorophyta Spirogyra maxima Cyanobacteria Entophysalis sp. Chlorophyta Spirogyra meinningensis Cyanobacteria Eucapsis sp. Chlorophyta Spirogyra notabilis Cyanobacteria Fischerella ambigua Chlorophyta Spirogyra Occidentalis Cyanobacteria Fischereia muscicoia Chlorophyta Spirogyra praiensis Cyanobacteria Fremyella diplosiphon Chlorophyta Spirogyra quadrilaminata Cyanobacteria Gloeocapsa alpicola Chlorophyta Spirogyra rhizobrachialis Cyanobacteria Gloeocapsa sp. Chlorophyta Spirogyra sp. Cyanobacteria Gioeotrichia echinitiata Chlorophyta Spirogyra varians Cyanobacteria Gloeotrichia ghosi Chlorophyta Stichococcus & Heterococcus spp. Cyanobacteria Gloeotrichia sp. Chlorophyta Stichococcus chodaii Cyanobacteria Hapaiosiphon welwitschii Chlorophyta Stichococcus fragilis Cyanobacteria Leptolyngbya nodulosa Chlorophyta Stichococcus mirabilis Cyanobacteria Lyngbya aestuarii Chlorophyta Stichococci is Sequoieti Cyanobacteria Lyngbya kuetzingii Chlorophyta Stigeoclonium aestivale Cyanobacteria Lyngbya lagerheinii Chlorophyta Stigeoclonium farctiin Cyanobacteria Lyngbya purptirem Chlorophyta Stigeoclonium pascheri Cyanobacteria Lyngbya sp. Chlorophyta Stigeoclonium subsectindum Cyanobacteria Mastigocladus iaminostis Chlorophyta Stigeoclonium tentie Cyanobacteria Merismopedia giatica f. insignis Chlorophyta Stigeoclonium variabile Cyanobacteria Merismopedia sp. Chlorophyta Tetradesmus cumbricus Cyanobacteria Microcoleus sp. Chlorophyta Zygnema annostin Cyanobacteria Microcoietis vaginati is var. cyano-viridis Chlorophyta Zygnema cylindricum Cyanobacteria Microcystis aeruginosa Chlorophyta Zygnema extentie Cyanobacteria Microcystis fios-aquae Chlorophyta Zygnema sp. Cyanobacteria Microcystis sp. Chlorophyta Zygnema spontanet in Cyanobacteria Nodularia harveyana Chlorophyta Zygnemasterile Cyanobacteria Nodularia sptimigena Cryptophyta Campylomonas reflexia Cyanobacteria Nostoc calcicoia Cryptophyta Chroomonas coerulea Cyanobacteria NoStoc commune Cryptophyta Chroomonas diplococca Cyanobacteria Nostocedaphicum Cryptophyta Chroomonas pochmani Cyanobacteria Nostocellipsosportin Cryptophyta Chroomonas sp. Cyanobacteria Nostocfoliaceum Cryptophyta Cryptochrysis sp. Cyanobacteria Nostoclongstafi Cryptophyta Cryptomonas ovata Cyanobacteria Nostoc parmeloides Cryptophyta Cryptomonas ovata var. palustris Cyanobacteria Nostoc piscinale Cryptophyta Cryptomonas Ozolini Cyanobacteria Nostoc punctiforme Cryptophyta Cryptomonas sp. Cyanobacteria Nostoc sp. Cryptophyta Hemiseimis sp. Cyanobacteria Nostoczetterstedtii Cryptophyta Proteomonas Suicata Cyanobacteria Osciliatoria amoena Cryptophyta Rhodomonas Saina Cyanobacteria Osciliatoria anima is US 2011/02751 18 A1 Nov. 10, 2011 23

TABLE 3-continued TABLE 3-continued

EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA AND, ORINTRA-CELLULAR CELLULASE ENZYMES AND, OR INTRA-CELLULAR CELLULASE ENZYMES ALGAE STRAINS ALGAE STRAINS

Division Strain Division Strain Cyanobac eria Osciliatoria borneti EuglenOZOa. Eugiena laciniata Cyanobac eria Osciliatoria brevis Euglenozoa Eugiena mutabilis Cyanobac eria Osciliatoria iud Euglenozoa Eugiena myxocylindracea Cyanobac eria Osciliatoria lutea Euglenozoa Eugiena piscifornis var. Obtusa Cyanobac eria Osciliatoria lutea war. contoria Euglenozoa Eugiena proxima Cyanobac eria Oscillatoria prolifera Euglenozoa Eugiena rubra Cyanobac eria Oscillatoria sp. Euglenozoa Eugiena Sanguinea Cyanobac eria Osciliatoria tentiis Euglenozoa Eugiena sp. Cyanobac eria Phormidium autumnaie Euglenozoa Eugiena spirogyra Cyanobac eria Phormidium boneri Euglenozoa Eugiena Stellata Cyanobac eria Phormidium foveolarum Euglenozoa Eugiena terricola Cyanobac eria Phormidium fragile Euglenozoa Eugiena tripteris Cyanobac eria Phormidium inundatum Euglenozoa Eutrepia pertyi Cyanobac eria Phormidium iuridium var. Oivace Euglenozoa Lepocincis buetschii Cyanobac eria Phormidium persicinum Euglenozoa Lepocincis ovata var. defiandriana Cyanobac eria Phormidium sp. Euglenozoa Phacus acuminata Cyanobac eria Plectonema bonyanum Euglenozoa Phacus brachykentron Cyanobac eria Plectonema sp. Euglenozoa Phacus caudata Cyanobac eria Pleurocapsa iiiiginosa Euglenozoa Phacus megalopsis Cyanobac eria Porphyrosiphon notarisii Euglenozoa Phacus pusillus Cyanobac eria Rubidibacteriacunae Euglenozoa Phacus triqueter Cyanobac eria Schizothrix calcicoia Euglenozoa Trachelomonas grandis Cyanobac eria Schizothrix calcicoia war. radiata Euglenozoa Trachelomonas hispida Cyanobac eria Schizothrix calcicola var. vermiformis Euglenozoa Trachelomonas hispida var. coronata Cyanobac eria Scytonema Euglenozoa Trachelomonas oblonga war. punctata Cyanobac eria Scytonema crispiim Euglenozoa Trachelomonas voivocina Cyanobac eria Scytonema hofmanni Euglenozoa Trachelomonas voivocinopsis var. spiralis Cyanobac eria Scytonema sp. Glaucophyta Cyanophora biloba Cyanobac eria Spirirestis rafaelensis Glaucophyta Cyanophora paradoxa Cyanobac eria Spiritiina major Glaucophyta Glaucocystis nostochinearlin Cyanobac eria Spiritiina maxima laptop nyla Calyptrosphaera sphaeroidea Cyanobac eria Spiritiina platensis ny Chrysochromulina brevifilum Cyanobac eria Spiritiina sp. ny Coccolithophora sp. Cyanobac eria Spiritiina Subsalsa ny Coccoithus neoheis Cyanobac eria Spirulina subsalsa f. versicolor ny Cricosphaera carterae Cyanobac eria Starria Zimbabweensis ny Dicrateria inornata Cyanobac eria Symphyonemopsis kainiensis ny Emiliania huxleyi Cyanobac eria Sympioca miscortin ny Isochrysis afgalbana Cyanobac eria Synechococcus ny Isochrysis galbania Cyanobac eria Synechococcus cedrorum ny Isochrysis sp. Cyanobac eria Synechococci is elongatus ny Ochrosphaera neapolitana Cyanobac eria Synechococci is sp. ny Ochrosphaera verrucosa Cyanobac eria Synechocystis nigrescens Pavlova gyrans Cyanobac eria Synechocystis sp. Paviova iutheri Cyanobac eria Tolypothrix distoria var. Sympiocoides Pseudoisochrysis paradoxia Dinophy Amphidinium carterae Sarcinochrysis marina Dinophy Amphidinium rhynchocephalum O O C O Asterosiphon dichotomats Dinophy Ceratocorys horrida Ooc Aureotimbra iagiinensis Dinophy Gyrodinium dorsum Ooc Bodaneia iauterborni Dinophy Heterocapsa niei Ooc Botrydiopsis a rhiza Dinophy Heterocapsa pigmeae Ooc Botrydium cystostin Dinophy Karenia brevis Ooc Bumilieria exis Dinophy Oxyrrhis marina Ooc Bumilieria Siculia Dinophy Peridinium foliaceum Ooc Bumilieriopsis sp. Dinophy Peridinium inconspicuum Ooc Chattonella japonica Dinophy Peridinium sociaie Ooc Chiorideia miniata Dinophy Prorocentrum cassubicum Ooc Chiorocioster Soiani Dinophy Protocentrum triestintin Ooc Chlorocioster sp. Dinophy Pyrocystis lunula Ooc Chromina nebulosa Dinophy Pyrocystis noctiluca Ooc Chrysochaete britannica Dinophy Scrippsiella trochoidea Ooc Dictyopteris repens Dinophyta Zooxantheia microadriatica Ooc Dictyota ciliiolata Euglenozo 8. Coiacium micronatum Ooc Dictyota dichotoma Euglenozo 8. Coiacium vesiculosum Ooc Dinobryon sp. Euglenozo 8. Eugiena actis var. gracilis Ooc Ectocarpus Silicatiosus Euglenozo 8. Eugiena anabaena Ooc Ectocarpus sp. Euglenozo 8. Eugiena Cantabrica Ooc Ectocarpus variabilis Euglenozo 8. Eugiena caudata Ooc Ellipsoidion sp. Euglenozo 8. Eugiena deses Ooc Epipyxis pitichra Euglenozo 8. Eugiena geniculata var. terricola Ooc O Eustigmatos magna US 2011/02751 18 A1 Nov. 10, 2011 24

TABLE 3-continued TABLE 3-continued

EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA- EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA AND, ORINTRA-CELLULAR CELLULASE ENZYMES AND, OR INTRA-CELLULAR CELLULASE ENZYMES ALGAE STRAINS ALGAE STRAINS

Division Strain Division Strain Oochrophyta Heterococci is caespitosus Rhodophyta Bostrychia tenella Oochrophyta Heterococcus cf. caespitosis Rhodophyta Botryocladia andreana Oochrophyta Heterococcus cf. endoithicus Rhodophyta Botryocladia boergesenii Oochrophyta Heterococcus cf. pleurococcoides Rhodophyta Botryocladia pyriformis Oochrophyta Heterococcus cf. proinematoides Rhodophyta Bryothamnion triqutrum Oochrophyta Heterococcus chodaii Rhodophyta Callithamnion baileyi Oochrophyta Heterococcus fuorinensis Rhodophyta Callithamnion byssoides Oochrophyta Heterococcits mainxi Rhodophyta Callithamnion corymbosum Oochrophyta Heterococcus moniiformis Rhodophyta Caithamnion haiae Oochrophyta Heterococci is protonematoides Rhodophyta Callithamnion paschale Oochrophyta Heterococci is sp. Rhodophyta Caithamnion roseum Oochrophyta Heterococcus sp. Pleuroscoccoides Rhodophyta Callithamnion sp. Oochrophyta Heterothrix debiis Rhodophyta Caloglossa intermedia Oochrophyta Heterotrichella gracilis Rhodophyta Caloglossa leprietiri f. pygmaea Oochrophyta Hibberdia magna Rhodophyta Ceranium sp. Oochrophyta Lagynion Scheifei Rhodophyta Champia parvula Oochrophyta Mallomonas as maindae Rhodophyta Chondrus crispus Oochrophyta Mischooocci is sphaerocephalus Rhodophyta Compsopogon coeruleus Oochrophyta Monodus subterraneus Rhodophyta Compsopogon hookeri Oochrophyta Nannochloropsis oculata Rhodophyta Compsopogon Oishi Oochrophyta Ochromonas sp. Rhodophyta Compsopogonopsis leptoclados Oochrophyta Ochromonas spherocystis Rhodophyta Chinagloia andersonii Oochrophyta Ophiocytium maius Rhodophyta Cyanidium caldarium Oochrophyta Phaeopiaca thallosa Rhodophyta Cystoclonium purpureum Oochrophyta Phaeoschizochlamys mucosa Rhodophyta Dasya pedicellata Oochrophyta Pleurochloris meiringensis Rhodophyta Dasya rigidia Oochrophyta Pseudobumilieriopsis pyrenoidosa Rhodophyta Digenea simplex Oochrophyta Sorocarpus tivaeformis Rhodophyta Dixoniella grisea Oochrophyta Spermatochnis paradoxus Rhodophyta Erythrocladia sp. Oochrophyta Sphacelaria cirrosa Rhodophyta Erythrotrichia carnea Oochrophyta Sphacellaria rigidula Rhodophyta Eupogodon plantis Oochrophyta Sphacellaria sp. Rhodophyta Flintiella sanguinaria Oochrophyta Stichogloea doederleinii Rhodophyta Geidiopsis intricata Oochrophyta Synura petersenii Rhodophyta Giaticosphaera vacuolata Oochrophyta Synura uveila Rhodophyta Graciliaria debilis Oochrophyta Tribonema missouriense Rhodophyta Graciliaria foliifera Oochrophyta Tribonema sp. Rhodophyta Graciliaria verrucosa Oochrophyta Vacuoiaria virescens Rhodophyta Grateloupia filicina Oochrophyta Vaticheria bursata Rhodophyta Griffithsia pacifica Oochrophyta Vaticheria geminata Rhodophyta Heterosiphonia plumosa Oochrophyta Vaticheria sessiis Rhodophyta Hildenbrandia prototypus Oochrophyta Vaticheria terrestris Rhodophyta Hildenbrandia riviliaris Oochrophyta Vischeria punctata Rhodophyta Hypnea muscifornis Rhodophyta Acrochaetium fiexuosum Rhodophyta Lomentaria articulata Rhodophyta Acrochaetium pectiniatum Rhodophyta Lomentaria Orcadensis Rhodophyta Acrochaetium plumosum Rhodophyta Lophocladia trichociados Rhodophyta Acrochaetium proskatieri Rhodophyta Nemation multifidum Rhodophyta Acrochaeti in Sagraeant in Rhodophyta Nemationopsis Shawi f. caroliniana Rhodophyta Acrochaetium sp Rhodophyta Nenaionopsis tortuosa Rhodophyta Acrosorium incinatin Rhodophyta Neoagardhiella baileyi Rhodophyta Anfractutofilum umbracolens Rhodophyta Palmaria palmata Rhodophyta Antithamnion defectum Rhodophyta Phyllophora membranacea Rhodophyta Antithamnion glanduliferum Rhodophyta Phyllophora truncata Rhodophyta Apoglosslim ruscifolium Rhodophyta Polyneura hilliae Rhodophyta Asterocytis ranosa Rhodophyta Polyneura latissima Rhodophyta Asterocytis sp. Rhodophyta Polysiphonia boldii Rhodophyta Atidotiinella eugenea Rhodophyta Polysiphonia echinata Rhodophyta Audouineia hermannii Rhodophyta Porphyra eucosticta Rhodophyta Bangia afisco-purpure Rhodophyta Pseudochantransia sp. Rhodophyta Bangia atro-purpurea Rhodophyta Pierociadia americana Rhodophyta Bangia fisco-purpurea Rhodophyta Pierociadia bartietti Rhodophyta Bangiopsis subsimplex Rhodophyta Pterocladia capillacea Rhodophyta Batrachospermum intortum Rhodophyta Ptilothamnion sp. Rhodophyta Batrachospermain macrosportin Rhodophyta Purpureofiliim apprenoidigerim Rhodophyta Batrachospermum moniiforme Rhodophyta Rhodeia maculata Rhodophyta Batrachospermum sirodotia Rhodophyta Rhodochaete parvula Rhodophyta Batrachospermum sp. Rhodophyta Rhodochorton purpureum Rhodophyta Batrachospermain vagiin var. keratophylum Rhodophyta Rhodochorion tennie Rhodophyta Boldia erythrosiphon Rhodophyta Rhodosorus marinus Rhodophyta Bostrychia bispora Rhodophyta Rhodospora Sordida US 2011/02751 18 A1 Nov. 10, 2011 25

TABLE 3-continued TABLE 4-continued

EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA- FURTHEREXAMPLES OF ALGAE STRAINS PRODUCING EXTRA AND, OR INTRA-CELLULAR CELLULASE ENZYMES AND, OR INTRA-CELLULAR CELLULASE ENZYMES ALGAE STRAINS ALGAE STRAINS Division Strain Division Genus specie Rhodophyta Rhodymenia cf. Ardisonnei Rard Cor Chlorophyta Chlorosarcinopsis gelatinosa Rhodophyta Rhodymenia pseudopalmata Chlorophyta Chlorosarcinopsis minuta Rhodophyta Seirospora griffithsiana Chlorophyta Choricystis sp. Rhodophyta Sirodotia sp. Chlorophyta Coelastropsis costata Rhodophyta Sirodotia Suecica Chlorophyta Coeiastrum astroideum Rhodophyta Sirodotia tentissina Chlorophyta Coelastrum microporum Rhodophyta Soieria tenera Chlorophyta Coeiastrum morus Rhodophyta Spermothamnion spelt incarnin Chlorophyta Coelastrum pseudomicroportin Rhodophyta Spermothamnion turneri Chlorophyta Coeiastrum reticulatum Rhodophyta Spyridia fillimentosa Chlorophyta Coenochloris pyrenoidosa Rhodophyta Stylonema alsidii Chlorophyta Coleochlamys cucumis Rhodophyta Thorea hispida Chlorophyta Cosmarium hoimiense Rhodophyta Thorea okaida Chlorophyta Cosmarium meneghini Rhodophyta Thorea riekei Chlorophyta Cosmarium subcrenaium Chlorophyta Crucigenia tetrapedia Rhodophyta Thorea violiacea Chlorophyta Crucigeniella pulchra Rhodophyta Trailieiia intricaia Chlorophyta Dictyococci is varians Rhodophyta Titoneva americana Chlorophyta Dictyosphaerium pulchelium Rhodophyta Tuomeya fluviatilis Chlorophyta Dictyosphaerium tetrachotomum Chlorophyta Dipiosphaera cf. chodatii Chlorophyta Enallax coelastroides Chlorophyta Enallax sp. TABLE 4 Chlorophyta Geminella sp. Chlorophyta Gonium pectorale FURTHEREXAMPLES OF ALGAE STRAINS PRODUCING EXTRA- Chlorophyta Graesiella vacuolata AND, OR INTRA-CELLULAR CELLULASE ENZYMES Chlorophyta Interfii in paradoxum ALGAE STRAINS Chlorophyta Kentrosphaera austriaca Chlorophyta Kentrosphaera gibberosa Division Genus specie Chlorophyta Keratococci is bicaudatus Chlorophyta Klebsormidium cf. Scopulinum Bacillariophyta Diades misgalica Chlorophyta Klebsormidium flaccidum Bacillariophyta Navicula atomits Chlorophyta Klebsormidium pseudostichococcus Chlorophyta Actinastrum hantzschii Chlorophyta Kiebsormidium rivuliare Chlorophyta Actinochloris sphaerica Chlorophyta Klebsormidium sp. Chlorophyta Ankistrodesmits spiralis Chlorophyta Koliella sempervirens Chlorophyta Apatococci is lobatus Chlorophyta Koliella spiculiformis Chlorophyta Asterarcy's cubensis Chlorophyta Lagerheinia marssonii Chlorophyta Auxenochiorella protothecoides Chlorophyta Lobosphaera sp. Chlorophyta Botryococci is protuberans Chlorophyta Macrochioris radiosa Chlorophyta Botryococci is stideticus Chlorophyta Monoraphidium arcuatum Chlorophyta Chaetophora cf. elegans Chlorophyta Monoraphidium cf. contortum Chlorophyta Chantransia sp. Chlorophyta Monoraphidium contortum Chlorophyta Characium sieboidii Chlorophyta Monoraphidium convolutum Chlorophyta Characium starri Chlorophyta Monoraphidium griffithii Chlorophyta Characium terrestre Chlorophyta Monoraphidium saxatile Chlorophyta Chlamydomonas actinochloris Chlorophyta Monoraphidium tortile Chlorophyta Chlamydomonas agregata Chlorophyta Mougeotia scalaris Chlorophyta Chlamydomonas augustae Chlorophyta Mougeotia sp. Chlorophyta Chlamydomonas cf. debaryana Chlorophyta Muriella sp. Chlorophyta Chlamydomonas cf. peterfii Chlorophyta Mychonastes sp. Chlorophyta Chlamydomonas cf. typica Chlorophyta Myrmecia bisecta Chlorophyta Chlamydomonas chlorococcoides Chlorophyta Natitococci is namniatus Chlorophyta Chlamydomonas dorsoventralis Chlorophyta Natitococci is sp. Chlorophyta Chlamydomonas geitieri Chlorophyta Neodesmus danubiais Chlorophyta Chlamydomonas macropy renoidosa Chlorophyta Neospongiococcim granaitin Chlorophyta Chlamydomonas moewusii Chlorophyta Nephrochlamys rotunda Chlorophyta Chlamydomonas nivais Chlorophyta Oocystis cf. nephrocytioides Chlorophyta Chlamydomonas peterfii Chlorophyta Oocystis lacustris Chlorophyta Chlamydomonas Segnis Chlorophyta Pediastrum biradiatum Chlorophyta Chlamydomonas subtilis Chlorophyta Pediastrum tetras Chlorophyta Chlorella cf. homosphaera Chlorophyta Pithophora roettieri Chlorophyta Chlorella homosphaera Chlorophyta Pleurastrum paucicellulare Chlorophyta Chiorelia kessieri Chlorophyta Pietrastrum sarcinoideum Chlorophyta Chiorelia mirabilis Chlorophyta Prasioiopsis ranosa Chlorophyta Chiorelia Sorokiniana Chlorophyta Protosiphon botryoides Chlorophyta Chlorokybus atmophyticus Chlorophyta Pseudendocionium basiiense Chlorophyta Chloromonas cf. paradoxia Chlorophyta Pseudendoclonium sp. Chlorophyta Chloromonas iemtlandica Chlorophyta Pseudococcomyxa cf. Simplex Chlorophyta Chioromonas rosae Chlorophyta Pseudococcomyxa simplex Chlorophyta Chlorosarcinopsis aggregata Chlorophyta Pseudococcomyxa sp. US 2011/02751 18 A1 Nov. 10, 2011 26

TABLE 4-continued TABLE 4-continued

FURTHEREXAMPLES OF ALGAE STRAINS PRODUCING EXTRA FURTHEREXAMPLES OF ALGAE STRAINS PRODUCING EXTRA AND, ORINTRA-CELLULAR CELLULASE ENZYMES AND, OR INTRA-CELLULAR CELLULASE ENZYMES ALGAE STRAINS ALGAE STRAINS

Division Genus specie Division Genus specie

OO hyta Raphidocelis inclinata not assigned to a phylum Desmodesmits fiscus OO hyta Raphidocelis subcapitata not assigned to a phylum Desmodesmus granitiatus OO hyta Raphidocelis valida not assigned to a phylum Desmodesmits hirsutius OO hyta Raphidonema Sempervirens not assigned to a phylum Desmodesmus quadricauda OO hyta Rhexinema paucicellularis not assigned to a phylum Desmodesmus Sempervirens OO hyta Rhopalocystis clictimis not assigned to a phylum Desmodesmits subspicatus OO hyta Scenedesmus cf. capitatus not assigned to a phylum Desmodesmus veilitaris OO hyta Scenedesmus cf. ecornis Ochrophyta Botrydiopsis alpina OO hyta Scenedesmus cf. pseudoarmaints Ochrophyta Bumilieriopsis filiformis OO hyta Scenedesmits incrassaiutius Ochrophyta Bumilieriopsis peterseniana OO hyta Scenedesmuspecsensis Ochrophyta Chloridella neglecta OO hyta Scenedesmits pleiomorphus Ochrophyta Chloridella simplex OO hyta Scenedesmits praetervisits Ochrophyta Chlorobotry's regularis OO hyta Schroederiella papillata Ochrophyta Ellipsoidion parvin OO hyta Scotiella chiorelioidea Ochrophyta Heterococcus breviceiuliaris OO hyta Scotiellopsis oocystifornis Ochrophyta Monodus guttula OO hyta Scotiellopsis reticulata Ochrophyta Monodus sp. OO hyta Scotiellopsis rubescens Ochrophyta Monodus subterraneus OO hyta Scotiellopsis terrestris Ochrophyta Nannochloropsis sp. OO hyta Selena Strain gracile Ochrophyta Nephrodieila minor OO hyta Seien.astrum rinoi Ochrophyta Pseudocharaciopsis ovalis OO hyta Sphaerocystis bilobata Ochrophyta Tribonema vulgare OO hyta Sphaerocystis Schroeteri Ochrophyta Vischeria helivetica OO hyta Spirogyra cf. Seniornata Ochrophyta Xanthonema bristoianum OO hyta Spirogyra communis Ochrophyta Xanthonema cf. debiis OO hyta Spirogyra lacustris Ochrophyta Xanthonema exiie OO hyta Spirogyra mirabilis Ochrophyta Xanthonema mucicoium OO hyta Spirogyra neglecta Ochrophyta Xanthonema sp. OO hyta Stichococcus cf. chiorelioides Prasinophyta Dunaieila bioculata OO hyta Stichococcus chioranthus Rhodophyta Microthamnion kuetzingianum OO hyta Stichococci is exiguus Rhodophyta Porphyridium aerugineum OO hyta Stichococcus minutus Rhodophyta Porphyridium purpureum OO hyta Stichococci is sp. Rhodophyta Porphyridium sordidum OO hyta Stigeoclonium helveticum Rhodophyta Porphyridium sp. OO hyta Stigeoclonium sp. OO hyta Tetradesmits wisconsinensis OO hyta Willea sp. orophyta Zygnema circumcarinatin 1. A method of producing fatty acids, comprising: hlorophyta Zygnema peiosportin Cyanobacteria Bracteacoccus minor (i) inoculating a mixture of at least one of cellulose, hemi Cyanobacteria Chlorococcum echinozygotum cellulose, and lignin with at least one microorganism Cyanobacteria Chlorococcum ellipsoideum strain and at least one algae strain, wherein said at least Cyanobacteria Chlorococcum hypnosporum one microorganism strain and said at least one algae Cyanobacteria Chlorococcum infusiorum Cyanobacteria Chiorococcum iobatum strain are aerobic and anaerobic organisms; Cyanobacteria Chiorococcum minutum (ii) growing said inoculated Strains under aerobic condi Cyanobacteria Chiorococcum scabelium tions, wherein: Cyanobacteria Chiorococcum vacuoiatum said at least one microorganism strain produces one or Cyanobacteria Chiopoietraedron bitridens Cyanobacteria Chiopoietraedron incus more cellulases, hemicellulases and laccases that hydro Cyanobacteria Chlorotetraedron polymorphium lyze at least one of cellulose, hemicellulose and lignin, Cyanobacteria Coccomyxa cf. gloeobotrydiformis to produce at least one of glucose, cellobiose, Xylose, Cyanobacteria Cocco myxa giaronensis Cyanobacteria Ettia carotinosa mannose, galactose, rhamnose, arabinose or other hemi Cyanobacteria Fortiea rugliosa cellulose Sugars in said mixture, and Cyanobacteria Neochioris biobata said at least one algae strain metabolizes acetic acid pro Cyanobacteria Neochioris texensis duced in a pretreatment step and also metabolizes said at Cyanobacteria Neochloris vigensis Cyanobacteria Spongiochloris spongiosa least one of glucose, cellobiose, Xylose, mannose, galac Cyanobacteria Tetraedron caudatum tose, rhamnose, arabinose or other hemicellulose Sugars Cyanobacteria Tetraedron minimum produced by said at least one microorganism strain; Cyanobacteria Tetrastrum komareki Euglenozoa Eugiena graciis var. trophora (iii) growing under anaerobic condition, and not assigned to a phylum Desmodesmits armaitis (a) either growing in heterotrophic condition, wherein: not assigned to a phylum Desmodesmus brasiliensis said at least one microorganism strain continues to pro not assigned to a phylum Desmodesmus cf. corailinus duce one or more cellulases, hemicellulases, and/or not assigned to a phylum Desmodesmus cf. gut winski not assigned to a phylum Desmodesmus cf. opoiensis var. mononensis laccases that hydrolyze at least one of cellulose, hemi not assigned to a phylum Desmodesmus cf. pannonicus cellulose, and lignin, and thereby produces at least not assigned to a phylum Desmodesmus cf. Spinostis one fermentation product comprising one or more alcohols in said mixture, and US 2011/02751 18 A1 Nov. 10, 2011 27

said at least one algae strain uses part of said at least one 15. The method of claim 14, wherein said extracellular of glucose, cellobiose, Xylose, mannose, galactose, and/or intracellular cellulase, hemicellulase, and/or laccase rhamnose, arabinose or other hemicellulose Sugars producer is a fungus or bacteria selected from the group produced by said at least one microorganism; consisting of Humicola, Trichoderma, Penicillium, Rumino (b) or growing in phototrophic condition, wherein: coccus, Bacillus, Cytophaga, Sporocytophaga, Humicola said at least one microorganism strain continues to pro grisea, Trichoderma harzianum, Trichodermalignorum, Tri duce one or more cellulases, hemicellulases, and/or choderma reesei, Penicillium verruculosum, Ruminococcus laccases that hydrolyze at least one of cellulose, hemi albus, Bacillus subtilis, Bacillus thermoglucosidasius, cellulose, and lignin, and thereby produces at least Cytophaga spp., Sporocytophaga spp., and Fusarium one fermentation product comprising one or more Oxysporum. alcohols and CO in said mixture, and said at least one algae strain uses most of said CO, part 16. The method of claim 15, wherein said at least one orall of said at least one fermentation product and part microorganism Strain is a fungus or a bacteria. of said at least one of glucose, cellobiose, Xylose, 17. The method of claim 15, wherein said at least one mannose, galactose, rhamnose, arabinose or other microorganism Strain is Fusarium oxysporum. hemicellulose Sugars produced by said at least one 18. The method of claim 1, wherein said at least one micro microorganism; organism strain produces at least one fermentation product (iv) growing under aerobic conditions, wherein: selected from the group consisting of Acetic acid, Acetate, said at least one algae strain metabolizes said at least one Acetone, 2,3-Butanediol, Butanol, Butyrate, CO2, Ethanol, fermentation product produced in step (iii) to produce Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, one or more fatty acids, and Succinate, and other fermentation products. said at least one microorganism continues producing said 19. The method of claim 1, wherein said at least one micro one or more cellulases, hemicellulases, and/or laccases; organism strain has been evolutionarily modified to metabo and lize pretreated biomass targeted more efficiently. (v) optionally recovering said one or more fatty acids. 20. The method of claim 19, wherein said at least one 2. The method of claim 1, wherein said method is per evolutionarily modified microorganism strain produces one formed under one or more additional Successive het or more cellulases, hemicellulases and/orlaccases so that said erotrophic or phototrophic conditions. evolutionarily modified microorganism strain has greater 3. The method of claim 1, further comprising growing capacity to metabolize cellulose and hemicelluloses with lig under one or more additional successive aerobic and anaero nin as compared to the unmodified wild-type version of the bic conditions. microorganism. 4. The method of claim 1, wherein said at least one micro 21. The method of claim 1, wherein said at least one micro organism strain is evolved for tolerance to furfural and acetic organism strain has been evolutionarily modified by at least acid and said at least one algae strain is evolved for tolerance one method selected from the group consisting of serial trans to furfural. fer, serial dilution, genetic engine, continuous culture, and 5. The method of claim 1, wherein the mixture in step (i) chemostat. further comprises at least one of furfural and acetic acid. 22. The method of claim 21, wherein said method is con 6. The method of claim 1, wherein said method uses all or tinuous culture. part of said CO, so there is no or little residual CO released 23. The method of claim 19, wherein said at least one as a byproduct of said method. microorganism strain is Fusarium oxysporum and has been 7. The method of claim 1, wherein the mixture in step (i) is evolutionarily modified by continuous culture. obtained from a biomass. 24. The method of claim 1, wherein said at least one micro 8. The method of claim 7, wherein said biomass is a plant organism strain has been evolutionary modified for a specific biomass. biomass plant. 9. The method of claim 7, wherein said biomass is obtained from plant or animal waste. 25. The method of claim 1, wherein said one or more 10. The method of claim 8, wherein said plant biomass cellulases is at least one selected from the group consisting of undergoes pretreatment by acid hydrolysis and heat treatment endoglucanase, exoglucanase, and B-glucosidase, hemicellu to produce said mixture inoculated in step (i). lases and optionally laccase. 11. The method of claim 8, wherein said plant biomass 26. The method of claim 1, further comprising measuring comprises: cellulase and/or hemicellulase activity in step (ii) and/or the 5-35% lignin; amount of fermentation products in step (iii), and depending 10-35% hemicellulose; and on the quantity of said products in the Supernatant, proceed 10-60% cellulose. ing to the next step. 12. The method of claim 8, wherein said plant biomass is 27. The method of claim 1, wherein said at least one algae obtained from at least one selected from the group consisting strain is selected from the group consisting of green algae, red of Switchgrass, corn Stover, and mixed waste of plant. algae, blue-green algae, cyanobacteria and diatoms. 13. The method of claim 1, wherein said at least one micro 28. The method of claim 27, wherein said at least one algae organism strain is an extracellular and/or intracellular cellu strain is selected from the group consisting of Monalanthus lase, hemicellulase, and/or laccase enzyme producer micro Salina. Botryococcus Braunii, Chlorella prototecoides, Out organism. irococcus sp., Scenedesmus obliquus, Nannochloris sp.: 14. The method of claim 13, wherein said extracellular Dunaliella bardawil (D. Salina); Navicula pelliculosa, and/or intracellular cellulase, hemicellulase, and/or laccase Radiosphaera negevensis, Biddulphia aurita, Chlorella vul producer is selected from the group consisting of: garis, Nitzschia palea, Ochromonas dannica, Chrorella prokaryote, bacteria, archaea, eukaryote, yeast and fungi. pyrenoidosa, Peridinium cinctum, Neochloris Oleabundans, US 2011/02751 18 A1 Nov. 10, 2011 28

Oocystis polymorpha, Chrysochromulina spp.; Scenedesmus feedstock for hydrocracking in an oil refinery to produce one acutus, Scenedesmus spp., Chlorella minutissima, Prynne or more compounds selected from the group consisting of sium parvum, Navicula pelliculosa, Scenedesmus dimor octane, gasoline, petrol, kerosene, diesel and otherpetroleum phus, Scotiella sp., Chorella spp.; Euglena gracilis, and Por product as solvent, plastic, oil, grease and fibers. phyridium cruentum. 44. The method of claim 1, further comprising, after step 29. The method of claim 1, wherein said at least one algae (v), direct transesterification of cells of said at least one algae strain has been evolutionarily modified to metabolize said at strain to produce fatty acids for biodiesel fuel. least one fermentation product. 45. The method of claim 44, wherein the direct transesteri 30. The method of claim 1, wherein growth of said at least fication comprises breaking the algae cells, releasing fatty one algae Strain is not inhibited by the presence of one or more acids and transesterification through a base or acid method oflignin, furfural, salts, cellulase enzymes and hemicellulase with methanol or ethanol to produce biodiesel fuel. enzymes. 46. The method of claim 1, wherein said at least one algae 31. The method of claim 1, wherein said at least one algae strain is adapted to use waste glycerol, as carbon Source, strain can grow in one or more conditions selected from the produced by the transesterification reaction without pretreat group consisting of aerobic, anaerobic, phototrophic, and ment or refinement to produce fatty acids for biodiesel pro heterotrophic. duction. 47. A product comprising an isolated algae adapted to 32. The method of claim 29, wherein said at least one algae metabolize waste glycerol, wherein said adaptation does not strain has been evolutionarily modified to heterotrophically include genetic modification. and/or phototrophically metabolize as a carbon Source said at 48. A product comprising an isolated biomass-cell culture least one fermentation product and said at least one algae mixture under conditions comprising at least a plant biomass, strain can optionally metabolize as a carbon source soluble one microorganism adapted to saccharify said biomass and Sugars released by a pretreatment of the mixture prior to step one algae adapted to metabolize one product of said saccha (i). rification. 33. The method of claim 1, wherein said at least one algae 49. A product comprising an evolutionarily modified strain has been evolutionarily modified by at least one method microorganism (EMO) wherein said organism is adapted to selected from the group consisting of serial transfer, serial grow under culture conditions comprising the presence of dilution, genetic engine, continuous culture, and chemostat. furfural, acetic acid, phenolics, lignin, salts or combinations 34. The method of claim 33, wherein said method is con thereof. tinuous culture. 50. A method of producing a fuel comprising contacting a 35. The method of claim 33, wherein said at least one algae Jatropha byproduct with a heterotrophic algae under culture strain is Chlorella protothecoides which has been evolution conditions sufficient for said heterotrophic algae to process arily modified by the continuous culture method. said byproduct to produce said fuel. 36. The method of claim 1, wherein said at least one algae 51. The mixture of claim 48, wherein said biomass inocu strain further metabolizes at least one of glucose, cellobiose, lating comprises at least one of cellulose, hemicellulose, and Xylose, mannose, galactose, rhamnose, arabinose or other lignin. hemicellulose Sugars, and waste glycerol. 52. The product of claim 48, wherein said conditions com 37. The method of claim 1, wherein said at least one algae prise aerobic growth, anaerobic growth or both. strain uses acetic acid as a carbon Source. 53. The method of claim 50, wherein said conditions com 38. The method of claim 1, wherein said at least one algae prise aerobic growth, anaerobic growth or both. strain produces no inhibitory by-product that inhibits growth 54. The product of claim 48, wherein said microorganism of said algae. is adapted to produce a greater amount of one or more cellu 39. The method of claim 1, wherein said recovering step (v) lases, hemicellulases and laccases that hydrolyze at least one comprises at least one selected from the group consisting of of cellulose, hemicellulose and lignin, to produce at least one filtration-centrifugation, flocculation, Solvent extraction, of glucose, cellobiose, Xylose, mannose, galactose, rham ultrasonication, microwave, pressing, distillation, thermal nose, arabinose or other hemicellulose Sugars in said mixture, evaporation, homogenization, hydrocracking (fluid catalytic as compared to a wild type of said microorganism. cracking), and drying of said at least one algae strain contain 55. The product of claim 48, wherein said algae is capable ing fatty acids. of metabolizing acetic acid glucose, cellobiose, Xylose, man 40. The method of claim 1, wherein supernatant recovered nose, galactose, rhamnose, arabinose or other hemicellulose in step (V) can be reused. Sugars produced by said at least one microorganism strain. 41. The method of claim 1, wherein step (iv) further com 56. The product of claim 55, wherein said algae is capable prises culturing and growing said at least one algae Strain of metabolizing C5 and C6 Sugars. under conditions for extracellular and/or intracellular produc 57. The product of claim 55, wherein said algae strain is tion of at least one compound selected from the group con further adapted to utilize substantially all of CO produced by sisting offatty acids, hydrocarbons, proteins, pigments, Sug said microoganism. ars, such as polysaccharides and monosaccharides, and 58. The product of claim 54, wherein said microorganism glycerol. is Fusarium oxysporum. 42. The method of claim 41, wherein said at least one 59. The method of claim 50, wherein said algae is Chlorella compound can be used for biofuel, cosmetic, alimentary, protothecoides mechanical grease, pigmentation, and medical use produc 60. The product of claim 48, wherein said algae is Chlo tion. rella protothecoides. 43. The method of claim 1, wherein said at least one algae strain produces hydrocarbon chains which can be used as