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Title:
A METHOD OF PRODUCING FATTY ACIDS FOR BIOFUEL, BIODIESEL, AND OTHER VALUABLE CHEMICALS
Document Type and Number:
WIPO Patent Application WO/2010/042842
Kind Code:
A2
Abstract:
The present invention relates to a method of producing fatty acids, by inoculating a mixture of at least one of cellulose, hemicellulose, and lignin with a microorganism strain and an algae strain, and growing said inoculated strains under successive aerobic-heterotrophic and either anaerobic-phototrophic or anaerobic-heterotrophic conditions creating symbiosis between the strains. Under a first aerobic-heterotrophic condition, the microorganism strain produces extracellulases that hydrolyze cellulose, hemicellulose and lignin, to produce sugars, such as glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars that are metabolized by the algae strain which also metabolizes acetic acid, glucose and hemicellulose from pretreatment. Then, either under a subsequent anaerobic-heterotrophic condition, the microorganism uses cellulose and produces fermentation products, and the algae strain uses part of the released sugars and exhibits a slower growth rate, or under a further anaerobic-phototrophic condition, the microorganism uses cellulose and produces fermentation products and CO2, and the algae strain uses the CO2 and part of the released sugars and the at least one fermentation product. Under a further aerobic-heterotrophic condition, the algae strain uses the fermentation products produced by the microorganism strain in a previous anaerobic step to produce one or more fatty acids, and the microorganism strain continues to produce extracellulases. The microorganism and algae strains are evolved for tolerance to furfural. The fatty acids can optionally be recovered and used for production of biodiesel fuel.

Inventors:
DE CRECY, Eudes (6716 SW 100th Lane, Gainesville, Florida, 32608, US)
Application Number:
US2009/060199
Publication Date:
April 15, 2010
Filing Date:
October 09, 2009
Export Citation:
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Assignee:
DE CRECY, Eudes (6716 SW 100th Lane, Gainesville, Florida, 32608, US)
International Classes:
C12P7/64
Domestic Patent References:
2008-01-03
Foreign References:
US20080102503A12008-05-01
US20070048859A12007-03-01
Other References:
OLSSON L ET AL: "Fermentation of lignocellulosic hydrolysates for ethanol production" ENZYME AND MICROBIAL TECHNOLOGY, STONEHAM, MA, US LNKD- DOI:10.1016/0141-0229(95)00157-3, vol. 18, no. 5, 1 January 1996 (1996-01-01), pages 312-331, XP002312595 ISSN: 0141-0229
LI XIUFENG ET AL: "Large-scale biodiesel production from microalga Chlorella protothecoides through heterotropic Cultivation in bioreactors" BIOTECHNOLOGY AND BIOENGINEERING,, vol. 98, no. 4, 1 November 2007 (2007-11-01), pages 764-771, XP002583837
XU H ET AL: "High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters" JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL LNKD- DOI:10.1016/J.JBIOTEC.2006.05.002, vol. 126, no. 4, 1 December 2006 (2006-12-01), pages 499-507, XP024956582 ISSN: 0168-1656 [retrieved on 2006-12-01]
SINGH A ET AL: "DIRECT FERMENTATION OF CELLULOSIC MATERIALS BY FUSARIUM OXYSPORUM 841: ACETIC ACID/ETHANOL PRODUCTION AND TOLERANCE" JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, XX, XX, vol. 38, no. 3, 1 January 1992 (1992-01-01), pages 227-236, XP008060816
MOSIER N ET AL: "Features of promising technologies for pretreatment of lignocellulosic biomass" BIORESOURCE TECHNOLOGY, ELSEVIER BV, GB LNKD- DOI:10.1016/J.BIORTECH.2004.06.025, vol. 96, no. 6, 1 April 2005 (2005-04-01), pages 673-686, XP025313257 ISSN: 0960-8524 [retrieved on 2005-04-01]
DE CRÉCY E ET AL: "Development of a novel continuous culture device for experimental evolution of bacterial populations" APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE LNKD- DOI:10.1007/S00253-007-1168-5, vol. 77, no. 2, 26 September 2007 (2007-09-26), pages 489-496, XP019560698 ISSN: 1432-0614
O'REILLY A M ET AL: "Defined coimmobilization of mixed microorganism cultures" ENZYME AND MICROBIAL TECHNOLOGY, STONEHAM, MA, US, vol. 17, no. 7, 1 July 1995 (1995-07-01), pages 636-646, XP002579233 ISSN: 0141-0229
HABIB A D ET AL: "Conversion of paper sludge into land application materials and diesel fuel precursors" COMPENDEX,, 1 January 1996 (1996-01-01), XP002554725
CHUNG Y-C ET AL: "Enzymatic saccharification and fermentation of xylose-optimized dilute acid-treated lignocellulosics" APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, THE HUMANA PRESS, INC, US LNKD- DOI:10.1385/ABAB:124:1-3:0947, vol. 124, no. 1-3, 1 March 2005 (2005-03-01), pages 947-962, XP002554724 ISSN: 0273-2289
Attorney, Agent or Firm:
JENSEN, Eric et al. (Young & Thompson, 209 Madison StreetSuite 50, Alexandria Virginia, 22314, US)
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Claims:
What we claim is;

1. A method of producing fatty acids, comprising:

(i) inoculating a mixture of at least one of cellulose, hemicellulose, and lignin with at least one microorganism strain and at least one algae strain, wherein said at least one microorganism strain and said at least one algae strain are aerobic and anaerobic organisms;

(ii) growing said inoculated strains under aerobic conditions, wherein: said at least one microorganism strain produces one or more cellulases, hemicellulases and laccases that hydrolyze at least one of cellulose, hemicellulose and lignin, to produce at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars in said mixture, and said at least one algae strain metabolizes acetic acid produced in a pretreatment step and also metabolizes said at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism strain;

(iii) growing under anaerobic condition, and

(a) either growing in heterotrophic condition, wherein: said at least one microorganism strain continues to produce one or more cellulases, hemicellulases, and/or laccases that hydrolyze at least one of cellulose, hemicellulose, and lignin, and thereby produces at least one fermentation product comprising one or more alcohols in said mixture, and said at least one algae strain uses part of said at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism;

(b) or growing in phototrophic condition, wherein: said at least one microorganism strain continues to produce one or more cellulases, hemicellulases, and/or laccases that hydrolyze at least one of cellulose, hemicellulose, and lignin, and thereby produces at least one fermentation product comprising one or more alcohols and CO2 in said mixture, and said at least one algae strain uses most of said CO2, part or all of said at least one fermentation product and part of said at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism; (iv) growing under aerobic conditions, wherein: said at least one algae strain metabolizes said at least one fermentation product produced in step (iii) to produce one or more fatty acids, and said at least one microorganism continues producing said one or more cellulases, hemicellulases, and/or laccases; and

(v) optionally recovering said one or more fatty acids.

2. The method of claim 1, wherein said method is performed under one or more additional successive heterotrophic or phototrophic conditions.

3. The method of claim 1 , further comprising growing under one or more additional successive aerobic and anaerobic conditions.

4. The method of claim 1, wherein said at least one microorganism strain is evolved for tolerance to furfural and acetic acid and said at least one algae strain is evolved for tolerance to furfural.

5. The method of claim 1, wherein the mixture in step (i) further comprises at least one of furfural and acetic acid.

6. The method of claim 1, wherein said method uses all or part of said CO2, so there is no or little residual CO2 released as a byproduct of said method.

7. The method of claim 1, wherein the mixture in step (i) is obtained from a biomass.

8. The method of claim 7, wherein said biomass is a plant biomass.

9. The method of claim 7, wherein said biomass is obtained from plant or animal waste.

10. The method of claim 8, wherein said plant biomass undergoes pretreatment by acid hydrolysis and heat treatment to produce said mixture inoculated in step (i).

11. The method of claim 8, wherein said plant biomass comprises: 5-35% lignin; 10-35% hemicellulose; and 10-60% cellulose.

12. The method of claim 8, wherein said plant biomass is obtained from at least one selected from the group consisting of: switchgrass, corn stover, and mixed waste of plant.

13. The method of claim 1, wherein said at least one microorganism strain is an extracellular and/or intracellular cellulase, hemicellulase, and/or laccase enzyme producer microorganism.

14. The method of claim 13, wherein said extracellular and/or intracellular cellulase, hemicellulase, and/or laccase producer is selected from the group consisting of: prokaryote, bacteria, archaea, eukaryote, yeast and fungi.

15. The method of claim 14, wherein said extracellular and/or intracellular cellulase, hemicellulase, and/or laccase producer is a fungus or bacteria selected from the group consisting of Humicola, Trichoderma, Penicillium, Ruminococcus , Bacillus, Cytophaga, Sporocytophaga, Humicola grisea, Trichoderma harzianum, Trichoderma lignorum, Trichoderma reesei, Penicillium verruculosum, Ruminococcus albus, Bacillus subtilis, Bacillus thermoglucosidasius , Cytophaga spp., Sporocytophaga spp., and Fusarium oxysporum.

16. The method of claim 15, wherein said at least one microorganism strain is a fungus or a bacteria.

17. The method of claim 15, wherein said at least one microorganism strain is

Fusarium oxysporum.

18. The method of claim 1, wherein said at least one microorganism strain produces at least one fermentation product selected from the group consisting of: Acetic acid, Acetate, Acetone, 2,3-Butanediol, Butanol, Butyrate, CO2, Ethanol, Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, Succinate, and other fermentation products.

19. The method of claim 1, wherein said at least one microorganism strain has been evolutionarily modified to metabolize pretreated biomass targeted more efficiently.

20. The method of claim 19, wherein said at least one evolutionarily modified microorganism strain produces one or more cellulases, hemicellulases and/or laccases so that said evolutionarily modified microorganism strain has greater capacity to metabolize cellulose and hemicelluloses with lignin as compared to the unmodified wild-type version of the microorganism.

21. The method of claim 1 , wherein said at least one microorganism strain has been evolutionarily modified by at least one method selected from the group consisting of serial transfer, serial dilution, genetic engine, continuous culture, and chemostat.

22. The method of claim 21, wherein said method is continuous culture.

23. The method of claim 19, wherein said at least one microorganism strain is Fusarium oxysporum and has been evolutionarily modified by continuous culture.

24. The method of claim 1 , wherein said at least one microorganism strain has been evolutionary modified for a specific biomass plant.

25. The method of claim 1, wherein said one or more cellulases is at least one selected from the group consisting of: endoglucanase, exoglucanase, and β-glucosidase, hemicellulases and optionally laccase.

26. The method of claim 1, further comprising measuring cellulase and/or hemicellulase activity in step (ii) and/or the amount of fermentation products in step (iii), and depending on the quantity of said products in the supernatant, proceeding to the next step.

27. The method of claim 1, wherein said at least one algae strain is selected from the group consisting of green algae, red algae, blue-green algae, cyanobacteria and diatoms.

28. The method of claim 27, wherein said at least one algae strain is selected from the group consisting of Monalanthus Salina; Botryococcus Braunii; Chlorella prototecoides; Outirococcus sp.; Scenedesmus obliquus; Nannochloris sp.; Dunaliella bardawil (D. Salina); Navicula pelliculosa; Radiosphaera negevensis; Biddulphia aurita; Chlorella vulgaris; Nitzschia palea; Ochromonas dannica; Chrorella pyrenoidosa; Peridinium cinctum; Neochloris oleabundans; Oocystis polymorpha; Chrysochromulina spp.; Scenedesmus acutus; Scenedesmus spp.; Chlorella minutissima; Prymnesium parvum; Navicula pelliculosa; Scenedesmus dimorphus; Scotiella sp.; Chorella spp.; Euglena gracilis; and Porphyridium cruentum.

29. The method of claim 1, wherein said at least one algae strain has been evolutionarily modified to metabolize said at least one fermentation product.

30. The method of claim 1, wherein growth of said at least one algae strain is not inhibited by the presence of one or more of lignin, furfural, salts, cellulase enzymes and hemicellulase enzymes.

31. The method of claim 1, wherein said at least one algae strain can grow in one or more conditions selected from the group consisting of: aerobic, anaerobic, phototrophic, and heterotrophic.

32. The method of claim 29, wherein said at least one algae strain has been evolutionarily modified to heterotrophically and/or phototrophicaly metabolize as a carbon source said at least one fermentation product and said at least one algae strain can optionally metabolize as a carbon source soluble sugars released by a pretreatment of the mixture prior to step (i).

33. The method of claim 1, wherein said at least one algae strain has been evolutionarily modified by at least one method selected from the group consisting of serial transfer, serial dilution, genetic engine, continuous culture, and chemostat.

34. The method of claim 33, wherein said method is continuous culture.

35. The method of claim 33, wherein said at least one algae strain is Chlorella protothecoides which has been evolutionarily modified by the continuous culture method.

36. The method of claim 1, wherein said at least one algae strain further metabolizes at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars, and waste glycerol.

37. The method of claim 1, wherein said at least one algae strain uses acetic acid as a carbon source.

38. The method of claim 1, wherein said at least one algae strain produces no inhibitory by-product that inhibits growth of said algae.

39. The method of claim 1, wherein said recovering step (v) comprises at least one selected from the group consisting of filtration-centrifugation, flocculation, solvent extraction, ultrasonication, microwave, pressing, distillation, thermal evaporation, homogenization, hydrocracking (fluid catalytic cracking), and drying of said at least one algae strain containing fatty acids.

40. The method of claim 1 , wherein supernatant recovered in step (v) can be reused.

41. The method of claim 1 , wherein step (iv) further comprises culturing and growing said at least one algae strain under conditions for extracellular and/or intracellular production of at least one compound selected from the group consisting of fatty acids, hydrocarbons, proteins, pigments, sugars, such as polysaccharides and monosaccharides, and glycerol.

42. The method of claim 41, wherein said at least one compound can be used for biofuel, cosmetic, alimentary, mechanical grease, pigmentation, and medical use production.

43. The method of claim 1, wherein said at least one algae strain produces hydrocarbon chains which can be used as feedstock for hydrocracking in an oil refinery to produce one or more compounds selected from the group consisting of octane, gasoline, petrol, kerosene, diesel and other petroleum product as solvent, plastic, oil, grease and fibers.

44. The method of claim 1, further comprising, after step (v), direct transesterification of cells of said at least one algae strain to produce fatty acids for biodiesel fuel.

45. The method of claim 44, wherein the direct transesterification comprises breaking the algae cells, releasing fatty acids and transesterification through a base or acid method with methanol or ethanol to produce biodiesel fuel.

46. The method of claim 1, wherein said at least one algae strain is adapted to use waste glycerol, as carbon source, produced by the transesterification reaction without pretreatment or refinement to produce fatty acids for biodiesel production.

47. A product comprising an isolated algae adapted to metabolize waste glycerol, wherein said adaptation does not include genetic modification.

48. A product comprising an isolated biomass-cell culture mixture under conditions comprising at least a plant biomass, one microorganism adapted to saccharify said biomass and one algae adapted to metabolize one product of said saccharification.

49. A product comprising an evolutionarily modified microorganism (EMO) wherein said organism is adapted to grow under culture conditions comprising the presence of furfural, acetic acid, phenolics, lignin, salts or combinations thereof.

50. A method of producing a fuel comprising contacting a Jatropha byproduct with a heterotrophic algae under culture conditions sufficient for said heterotrophic algae to process said byproduct to produce said fuel.

51. The mixture of claim 48, wherein said biomass inoculating comprises at least one of cellulose, hemicellulose, and lignin.

52. The product of claims 48 or 49, wherein said conditions comprise aerobic growth, anaerobic growth or both.

53. The method of claim 50, wherein said conditions comprise aerobic growth, anaerobic growth or both.

54. The product of claims 48 or 49, wherein said microorganism is adapted to produce a greater amount of one or more cellulases, hemicellulases and laccases that hydrolyze at least one of cellulose, hemicellulose and lignin, to produce at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars in said mixture, as compared to a wild type of said microorganism.

55. The product of claims 47, 48 or 50, wherein said algae is capable of metabolizing acetic acid glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism strain.

56. The product of claim 55, wherein said algae is capable of metabolizing C5 and C6 sugars.

57. The product of claim 55, wherein said algae strain is further adapted to utilize substantially all of CO2 produced by said microoganism.

58. The product of claim 54, wherein said microorganism is Fusarium oxysporum.

59. The method of claim 50, wherein said algae is Chlorella protothecoides

60. The product of claims 47, 48 or 49, wherein said algae is Chlorella protothecoides.

Description:
A METHOD OF PRODUCING FATTY ACIDS FOR BIOFUEL, BIODIESEL, AND OTHER VALUABLE CHEMICALS

BACKGROUND OF THE INVENTION

Petroleum is a non-renewable resource. As a result, many people are worried about the eventual depletion of petroleum reserves in the future. World petroleum resources have even been predicted by some to run out by the 21 st century (Kerr RA, Science 1998, 281, 1128).

This has fostered the expansion of alternative hydrocarbon products such as ethanol or other microbial fermentation products from plant derived feed stock and waste. In fact, current studies estimate that the United States could easily produce 1 billion dry tons of biomass (biomass feedstock) material (over half of which is waste) per year. This is primarily in the form of cellulosic biomass.

Cellulose is contained in nearly every natural, free-growing plant, tree, and bush, in meadows, forests, and fields all over the world without agricultural effort or cost needed to make it grow.

It is estimated that these cellulosic materials could be used to produce enough ethanol to replace 30% or more of the US energy needs in 2030. The great advantage of this strategy is that cellulose is the most abundant and renewable carbon source on earth and its efficient transformation into a useable fuel could solve the world's energy problem.

Cellulosic ethanol has been researched extensively. Cellulosic ethanol is chemically identical to ethanol from other sources, such as corn starch or sugar, but has the advantage that the cellulosic materials are highly abundant and diverse. However, it differs in that it requires a greater amount of processing to make the sugar monomers available to the microorganisms that are typically used to produce ethanol by fermentation.

Although cellulose is an abundant plant material resource, its rigid structure makes cellulose a difficult starting material to process. As a result, an effective pretreatment is needed to liberate the cellulose from the lignin seal and its crystalline structure so as to render it accessible for a subsequent hydrolysis step. By far, most pretreatments are done through physical or chemical means. In order to achieve higher efficiency, some researchers seek to incorporate both effects.

To date, the available pretreatment techniques include acid hydrolysis, steam explosion, ammonia fiber expansion, alkaline wet oxidation and ozone pretreatment. Besides effective cellulose liberation, an ideal pretreatment has to minimize the formation of degradation products because of their inhibitory effects on subsequent hydrolysis and fermentation processes.

The presence of inhibitors makes it more difficult to produce ethanol. Even though pretreatment by acid hydrolysis is probably the oldest and most studied pretreatment technique, it produces several potent inhibitors including furfural and hydroxymethyl furfural (HMF) which are by far regarded as the most toxic inhibitors present in lignocellulosic hydrolysate.

The cellulose molecules are composed of long chains of sugar molecules of various kinds. In the hydrolysis process, these chains are broken down to free the sugar, before it is fermented for alcohol production.

There are two major cellulose hydrolysis processes: i) a chemical reaction using acids, or an ii) an enzymatic reaction. However, current hydrolysis processes are expensive and inefficient. For example, enzymatic hydrolysis processes require obtaining costly cellulase enzymes from outside suppliers.

A further problem in transforming cellulosic products into ethanol is that up to 50% of the available carbon to carbon dioxide is inherently lost through the fermentation process. In addition, ethanol is more corrosive than gas and diesel. As a result, it requires a distinct distribution infrastructure as well as specifically designed engines. Finally, ethanol is 20-30% less efficient than fossil gas and as ethanol evaporates more easily, a higher percentage is lost along the whole production and distribution process.

A process that could produce biodiesel from cellulose would alleviate the problems associated with ethanol and other biodiesel productions.

Biodiesel obtained from microorganisms (e.g., algae and bacteria) is also non-toxic, biodegradable and free of sulfur. As most of the carbon dioxide released from burning biodiesel is recycled from what was absorbed during the growth of the microorganisms (e.g., algae and bacteria), it is believed that the burning of biodiesel releases less carbon dioxide than from the burning of petroleum, which releases carbon dioxide from a source that has been previously stored within the earth for centuries. Thus, utilizing microorganisms for the production of biodiesel may result in lower greenhouse gases such as carbon dioxide.

Some species of microorganisms are ideally suited for biodiesel production due to their high oil content. Certain microorganisms contain lipids and/or other desirable hydrocarbon compounds as membrane components, storage products, metabolites and sources of energy. The percentages in which the lipids, hydrocarbon compounds and fatty acids are expressed in the microorganism will vary depending on the type of microorganism that is grown. However, some strains have been discovered where up to 90% of their overall mass contain lipids, fatty acids and other desirable hydrocarbon compounds (e.g., Botryococcus).

Algae such as Chlorela sp. and Dunaliella are a source of fatty acids for biodiesel that has been recognized for a long time. Indeed, these eukaryotic microbes produce a high yield of fatty acids (20-80% of dry weight), and can utilize CO 2 as carbon with a solar energy source.

However, the photosynthetic process is not efficient enough to allow this process to become a cost effective biodiesel source. An alternative was to use the organoheterotrophic properties of Algae and have them grow on carbon sources such as glucose. In these conditions, the fatty acid yield is extremely high and the fatty acids are of a high quality. The rest of the dry weight is mainly constituted of proteins. However, the carbon sources used are too rare and expensive to achieve any commercial viability.

Lipid and other desirable hydrocarbon compound accumulation in microorganisms can occur during periods of environmental stress, including growth under nutrient-deficient conditions. Accordingly, the lipid and fatty acid contents of microorganisms may vary in accordance with culture conditions.

The naturally occurring lipids and other hydrocarbon compounds in these microorganisms can be isolated transesterified to obtain a biodiesel. The transesterification of a lipid with a monohydric alcohol, in most cases methanol, yields alkyl esters, which are the primary component of biodiesel.

The transesterification reaction of a lipid leads to a biodiesel fuel having a similar fatty acid profile as that of the initial lipid that was used (e.g., the lipid may be obtained from animal or plant sources). As the fatty acid profile of the resulting biodiesel will vary depending on the source of the lipid, the type of alkyl esters that are produced from a transesterification reaction will also vary. As a result, the properties of the biodiesel may also vary depending on the source of the lipid. (e.g., see Schuchardt, et al, TRANSESTERIFICATION OF VEGETABLE OILS: A REVIEW, J. Braz. Chem. Soc, vol. 9, 1, 199-210, 1998 and G. Knothe, FUEL PROCESSING TECHNOLOGY, 86, 1059-1070 (2005), each incorporated herein by reference).

SUMMARY

The present invention relates to a method for producing fatty acids from biomass, and in particular a method of producing fatty acids from biomass and for producing a biofuel from said fatty acids. In particular, the present invention relates to a method of producing fatty acids, by inoculating a biomass mixture of at least one of cellulose, hemicellulose, and lignin with a microorganism strain and an algae strain, that are both aerobic and anaerobic, and then growing said inoculated strains under heterotrophic condition and along successive aerobic and anaerobic conditions, or growing said inoculated strains under successive aerobic -heterotrophic and anaerobic -phototrophic conditions, creating symbiosis between the strains.

In the first case, under a first aerobic condition, the microorganism strain produces extracellulases that can hydrolyze cellulose, hemicellulose and lignin, to produce sugars, such as glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars, that can be metabolized by the algae strain which also can metabolize acetic acid from pretreatment. Under a subsequent anaerobic condition, the microorganism strain can use cellulose and can produce fermentation products, and the algae strain can use part of the released sugars and may exhibit a slower growth rate. Under a further aerobic condition, the algae strain can use the fermentation products produced by the microorganism strain in the previous anaerobic step and the algae can produce one or more fatty acids that can then be recovered, and the microorganism strain continues to produce extracellulases.

In the second case, under a first aerobic-heterotrophic condition, the microorganism strain produces extracellulases that can hydrolyze cellulose, hemicellulose and lignin, to produce sugars, such as glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars that can be metabolized by the algae strain which also can metabolize acetic acid, glucose and hemicellulose from a pretreatment. Then, under a subsequent anaerobic -phototrophic condition, the microorganism can use cellulose and can produce fermentation products and CO 2 , and the algae strain can use CO 2 and part of the released sugars and the at least one fermentation product. Under a further aerobic- heterotrophic condition, the algae strain can use the fermentation products produced by the microorganism strain to produce one or more fatty acids, and the microorganism strain continues to produce extracellulases.

In both cases, the microorganism and algae strains are evolved for tolerance to furfural and acetic acid.

The microorganism and algae strains are both aerobic and anaerobic.

The invention relates to symbiotic relationship between the microorganism strain and the algae strain during growth under alternating environmental conditions: either alternating aerobic-heterotrophic and anaerobic -heterotrophic conditions or alternating aerobic - heterotrophic and anaerobic -phototrophic conditions.

The recovered fatty acids can be used to produce biofuels, e.g., biodiesel.

The invention eliminates the need for costly enzymes produced by outside manufacturers that are required in conventional processes for bio-ethanol production. Also, no detoxification step is required in the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1. is a flowchart illustrating a conventional process for bio-ethanol production.

Fig 2. is a flowchart illustrating the general process for fatty acid production, alcohol production, and biofuel production according to an embodiement of the invention.

Fig 3. is a flowchart illustrating a specific process for fatty acid production, alcohol production, and biofuel production according to an embodiement of the invention, further depicting how the process eliminates the need for detoxification, the need for supplying outside enzymes as required in the conventional process for bio-ethanol production, and depicts how the process of the invention can be used to reduce carbon dioxide production.

Fig 4. is a flowchart illustrating a preferred embodiment of a specific process for fatty acid production, alcohol production, and biofuel production according to a preferred embodiment of the invention.

Fig 5. is a flowchart illustrating a preferred embodiment of a specific process for fatty acid production, alcohol production, CO 2 production and biofuel production according to a preferred embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to embodiments of the invention. Examples of embodiments are illustrated in the accompanying drawings. While the invention will be described in conjunction with these embodiments, it will be understood that it is not intended to limit the invention to such embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present invention.

The present invention relates to a method for producing fatty acids for possible use in biofuel production and alcohol production from biomass material. The method involves producing fatty acids, by inoculating a biomass mixture of at least one of cellulose, hemicellulose, and lignin with a microorganism strain and an algae strain, that are both aerobic and anaerobic, and then growing said inoculated strains under heterotrophic condition and along successive aerobic and anaerobic conditions, or growing said inoculated strains under successive aerobic -heterotrophic and anaerobic -phototrophic conditions, creating symbiosis between the strains.

In the first case, under a first aerobic condition, the microorganism strain produces extracellulases that hydrolyze cellulose, hemicellulose and lignin, to produce sugars, such as glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars, that are metabolized by the algae strain which also metabolizes acetic acid from pretreatment. Under a subsequent anaerobic condition, the microorganism strain uses cellulose and produces fermentation products, and the algae strain uses part of the released sugars and exhibits a slower growth rate. Under a further aerobic condition, the algae strain uses the fermentation products produced by the microorganism strain in the previous anaerobic step and the algae produces one or more fatty acids that are then recovered, and the microorganism strain continues to produce extracellulases.

In the second case, under a first aerobic-heterotrophic condition, the microorganism strain produces extracellulases that hydrolyze cellulose, hemicellulose and lignin, to produce sugars, such as glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars that are metabolized by the algae strain which also metabolizes acetic acid, glucose and hemicellulose from a pretreatment. Then, under a subsequent anaerobic - phototrophic condition, the microorganism uses cellulose and produces fermentation products and CO 2 , and the algae strain uses CO 2 and part of the released sugars and the at least one fermentation product. Under a further aerobic-heterotrophic condition, the algae strain uses the fermentation products produced by the microorganism strain to produce one or more fatty acids, and the microorganism strain continues to produce extracellulases.

The recovered fatty acids can be used to produce biofuels, e.g., biodiesel.

The microorganism and algae strains are pre-adapted/evolved to a pretreated medium resulting in tolerance to furfural and acetic acid.

More specifically, the invention is directed to a method of producing fatty acids, by: (i) inoculating a mixture of at least one of cellulose, hemicellulose, and lignin with at least one microorganism strain and at least one algae strain, wherein said at least one microorganism strain and said at least one algae strain are aerobic and anaerobic organisms;

(ii) growing said inoculated strains under aerobic and heterotrophic conditions, wherein: said at least one microorganism strain produces one or more cellulases, hemicellulases and laccases that hydrolyze at least one of cellulose, hemicellulose and lignin, to produce at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars in said mixture, and said at least one algae strain metabolizes acetic acid produced in a pretreatment step and also metabolizes said at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism strain, and;

(iii) growing under anaerobic and either heterotrophic or phototrophic condition, wherein: said at least one microorganism strain continues to produce one or more cellulases, hemicellulases, and/or laccases that hydrolyze at least one of cellulose, hemicellulose, and lignin, and thereby produces at least one fermentation product comprising one or more alcohols in whatever heterotrophic or phototrophic condition, and also CO 2 when in phototrophic condition, in said mixture, and said at least one algae strain uses CO2, part of said at least one fermentation product and part of said at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism, when in phototrophic environment, or said algae strain uses part of said at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism, when in heterotrophic condition;

(iv) growing under aerobic and heteroptrophic conditions, wherein: said at least one algae strain metabolizes said at least one fermentation product produced in step (iii) to produce one or more fatty acids, and said at least one microorganism continues producing said one or more cellulases, hemicellulases, and/or laccases; and

(v) optionally recovering said one or more fatty acids. In one embodiment, the method is performed under heterotrophic conditions. In another embodiment, the method involves further growing under one or more additional successive aerobic and anaerobic conditions.

In one embodiment, the method of the invention does not involve agitation of the mixture during said anaerobic conditions. In another embodiment, the invention there is optional agitation during said aerobic conditions. In another embodiment, the method involves further growing under one or more additional successive aerobic -heterotrophic and anaerobic -phototrophic conditions.

In a further embodiment, the method method uses all of the CO 2 , so there is no residual CO 2 released as a byproduct of the method of the invention.

In one embodiment, the microorganism strain is evolved for tolerance to furfural and acetic acid, and the algae strain is evolved for tolerance to furfural.

The mixture in step (i) can be obtained from biomass. Biomass is any organic material made from plants or animals, including living or recently dead biological material, which can be used as fuel or for industrial production. Most commonly, biomass refers to plant matter grown for use as biofuel, but it also includes plant or animal matter used for production of fibers, chemicals or heat. Biomass is a renewable energy source.

There are a wide variety of sources of biomass, including tree and grass crops and forestry, agricultural, and urban wastes, all of which can be utilized in the present invention. Examples of domestic biomass resources include agricultural and forestry residues, municipal solid wastes, industrial wastes, and terrestrial and aquatic crops.

There are many types of plants in the world, and many ways they can be used for energy production. In general there are two approaches: growing plants specifically for energy use, and using the residues from plants that are used for other things. The type of plant utilized in the present invention varies from region to region according to climate, soils, geography, population, and so on.

Energy crops (also called "power crops") can be grown on farms in potentially very large quantities. Trees and grasses, including those native to a region, are preferred energy crops, but other, less agriculturally sustainable crops, including corn can also be used.

Trees are a good renewable source of biomass for processing in the present invention. In addition to growing very fast, certain trees will grow back after being cut off close to the ground (called "coppicing"). This allows trees to be harvested every three to eight years for 20 or 30 years before replanting. Such trees (also called "short-rotation woody crops") grow as much as 40 feet high in the years between harvests. In cooler, wetter regions of the northern United States, varieties of poplar, maple, black locust, and willow are preferred. In the warmer Southeast, sycamore and sweetgum are preferred. While in the warmest parts of Florida and California, eucalyptus is likely to grow well.

Grasses are a good renewable source of biomass for use in the present invention. Thin-stemmed perennial grasses are common throughout the United States. Examples include switchgrass, big bluestem, and other native varieties, which grow quickly in many parts of the country, and can be harvested for up to 10 years before replanting. Thick-stemmed perennials including sugar cane and elephant grass can be grown in hot and wet climates like those of Florida and Hawaii. Annuals, such as corn and sorghum, are another type of grass commonly grown for food.

Oil plants are also a good source of biomass for use in the present invention. Such plants include, for example, soybeans and sunflowers that produce oil, which can be used to make biofuels. Another different type of oil crop is microalgae. These tiny aquatic plants have the potential to grow extremely fast in the hot, shallow, saline water found in some lakes in the desert Southwest.

In this regard, biomass is typically obtained from waste products of the forestry, agricultural and manufacturing industries, which generate plant and animal waste in large quantities.

Forestry wastes are currently a large source of heat and electricity, as lumber, pulp, and paper mills use them to power their factories. Another large source of wood waste is tree tops and branches normally left behind in the forest after timber-harvesting operations.

Other sources of wood waste include sawdust and bark from sawmills, shavings produced during the manufacture of furniture, and organic sludge (or "liquor") from pulp and paper mills.

As with the forestry industry, a large volume of crop residue remains in the field after harvest. Such waste could be collected for biofuel production. Animal farms produce many "wet wastes" in the form of manure. Such waste can be collected and used by the present invention to produce fatty acids for biofuel production.

People generate biomass wastes in many forms, including "urban wood waste" (such as shipping pallets and leftover construction wood), the biodegradable portion of garbage (paper, food, leather, yard waste, etc.) and the gas given off by landfills when waste decomposes. Even our sewage can be used as energy; some sewage treatment plants capture the methane given off by sewage and burn it for heat and power, reducing air pollution and emissions of global warming gases. In one embodiment, the present invention utilizes biomass obtained from plants or animals. Such biomass material can be in any form, including for example, chipped feedstock, plant waste, animal waste, etc.

Such plant biomass typically comprises: 5-35% lignin; 10-35% hemicellulose; and 10-60% cellulose.

The plant biomass that can be utilized in the present invention include at least one member selected from the group consisting of wood, paper, straw, leaves, prunings, grass, including switchgrass, miscanthus, hemp, vegetable pulp, corn, corn stover, sugarcane, sugar beets, sorghum, cassava, poplar, willow, potato waste, bagasse, sawdust, and mixed waste of plant, oil palm (palm oil) and forest mill waste.

In one embodiment of the invention, the plant biomass is obtained from at least one plant selected from the group consisting of: switchgrass, corn stover, and mixed waste of plant. In another embodiment, the plant biomass is obtained from switchgrass, due to its high levels of cellulose.

It should be noted that any such biomass material can by utilized in the method of the present invention.

The plant biomass can initially undergo a pretreatment to prepare the mixture utilized in step (i). Pretreatment is used to alter the biomass macroscopic and microscopic size and structure, as well as submicroscopic chemical composition and structure, so hydrolysis of the carbohydrate fraction to monomeric sugars can be achieved more rapidly and with greater yields. Common pretreatment procedures are disclosed in Nathan Mosier, Charles Wyman, Bruce Dale, Richard Elander, Y.Y. Lee, Mark Holtzapple, Michael Ladisch, "Features of promising technologies for pretreatment of lignocellulosic biomass," Bioresource Technology: 96, pp. 673-686 (2005), herein incorporated by reference, and discussed below.

Pretreatment methods are either physical or chemical. Some methods incorporate both effects (McMillan, 1994; Hsu, 1996). For the purposes of classification, steam and water are excluded from being considered chemical agents for pretreatment since extraneous chemicals are not added to the biomass. Physical pretreatment methods include comminution (mechanical reduction in biomass particulate size), steam explosion, and hydrothermolysis. Comminution, including dry, wet, and vibratory ball milling (Millett et al, 1979; Rivers and Emert, 1987; Sidiras and Koukios, 1989), and compression milling (Tassinari et al., 1980, 1982) is sometimes needed to make material handling easier through subsequent processing steps. Acids or bases could promote hydrolysis and improve the yield of glucose recovery from cellulose by removing hemicelluloses or lignin during pretreatment. Commonly used acid and base include, for example, H 2 SO 4 and NaOH, respectively. Cellulose solvents are another type of chemical additive. Solvents that dissolve cellulose in bagasse, cornstalks, tall fescue, and orchard grass resulted in 90% conversion of cellulose to glucose (Ladisch ct al, 1978; Hamilton ct al., 1984) and showed enzyme hydrolysis could be greatly enhanced when the biomass structure is disrupted before hydrolysis. Alkaline H 2 O 2 , ozone, organosolv (uses Lewis acids, FeCl3, (Al) 2 SO 4 in aqueous alcohols), glycerol, dioxane, phenol, or ethylene glycol are among solvents known to disrupt cellulose structure and promote hydrolysis (Wood and Saddler, 1988). Concentrated mineral acids (H 2 SO 4 , HCl), ammonia-based solvents (NH 3 , hydrazine), aprotic solvents (DMSO), metal complexes (ferric sodium tartrate, cadoxen, and cuoxan), and wet oxidation also reduces cellulose crystallinity and disrupt the association of lignin with cellulose, as well as dissolve hemicellulose. These methods, while effective, are too expensive for now to be practical when measured against the value of the glucose (approximately 5ø/lb). The following pretreatment methods of steam explosion, liquid hot water, dilute acid, lime, and ammonia pretreatments (AFEX), could have potential as cost-effective pretreatments.

It should be noted that any such pretreatment procedure can be utilized to alter the biomass to make the mixture utilized in the invention. In this regard, the microorganism in step (i) can be adapted to apply all pretreatment procedures and their associated residual compound that can include, for example, furfural, hydroxymethyl furfural(HMF), phenolics like 3,4-dihydroxybenzal-dehyde, 3 -methoxy-4-hydroxy -benzoic acid, cinnamic acid, anillin, vanillin alcohol, as well as sodium combinates like sodium hydroxide, nitrate combinates or ammonia, depending on the elected pretreatment method.

Acid pretreatment is a common pretreatment procedure. Acid pretreatment by acid hydrolysis and heat treatment can be utilized to produce the mixture inoculated in step (i) of the present invention. Any suitable acid can be used in this step, so long as the acid hydrolyzes hemicelluloses away from cellulose. Some common acids that can be used include a mineral acid selected from hydrochloric acid, phosphoric acid, sulfuric acid, or sulfurous acid. Sulfuric acid, for example, at concentration of about 0.5 to 2.0% is preferred. Suitable organic acids may be carbonic acid, tartaric acid, citric acid, glucuronic acid, acetic acid, formic acid, or similar mono- or polycarboxylic acids. The acid pretreatment also typically involves heating the mixture, for example, in a range of about 7O 0 C to 50O 0 C, or in a range of about 12O 0 C to 200 0 C, or in a range of 12O 0 C to 14O 0 C.

Such acid pretreatment procedure can be used to generate the mixture utilized in step

CO-

i i It should be noted that, when the biomass is obtained from plants, the mixture comprises at least one of cellulose, hemicellulose, lignin, furfural and acetic acid.

After the pretreatment procedure, the mixture in step (i) comprises at least one of cellulose, hemicellulose, and lignin. In step (i), this mixture is inoculated with at least one microorganism strain and at least one algae strain.

The strains are grown heterotrophically under alternating aerobic and anaerobic conditions or under successive aerobic-heterotrophic and anaerobic -phototrophic conditions.

To start, the strains are first grown under aerobic and heterotrophic conditions (step ii). Under aerobic and heterotrophic conditions, the microorganism strain produces one or more cellulases, hemicellulases, and/or laccases that hydrolyze at least one of cellulose, hemicellulose and lignin to produce at least one sugar, such as glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars in said mixture. Also, under the aerobic and heterotrophic conditions, the at least one algae strain metabolizes acetic acid, glucose and hemicellulose produced in a previous pretreatment step and also metabolizes one or more of the glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism strain, and produces fatty acids.

Then in step (iii), the mixture is grown under two possible anaerobic conditions: either heterotrophically or phototrophically. Under such anaerobic and heterotrophic conditions, the microorganism strain continues to produce cellulases, hemicellulases, and/or laccases that hydrolyze one or more of cellulose, hemicellulose, and lignin, and thereby produces at least one fermentation product comprising one or more alcohols. Also, under the anaerobic and heterotrophic conditions, the algae strain uses part of the sugars, i.e., glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism, thus producing one or more fatty acids. Otherwise, under anaerobic-phototrophic conditions, the microorganism strain continues to produce cellulases, hemicellulases, and/or laccases that hydrolyze one or more of cellulose, hemicellulose, and lignin, and thereby produces at least one fermentation product comprising one or more alcohols and CO 2 in said mixture. Also, under the anaerobic-phototrophic conditions, the at least one algae strain uses part or all of CO 2 , part or all of said at least one fermentation product and part of the sugars, i.e., glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by said at least one microorganism, thus producing one or more fatty acids. Then, in step (iv), the mixture is grown under a further aerobic and heterotrophic conditions, wherein said at least one algae strain metabolizes said at least one fermentation product produced in step (iii) to produce one or more fatty acids. Under this additional aerobic -heterotrophic condition, the at least one microorganism continues producing one or more cellulases, hemicellulases, and/or laccases.

In optional step (v), the one or more fatty acids are recovered.

Again, in one embodiment, the method is performed under heterotrophic conditions.

Also, the method comprises growing under one or more successive aerobic and anaerobic conditions.

Again, in one embodiment, the method of the invention does not involve agitation of the mixture during said anaerobic conditions. In another embodiment, the invention involves optional agitation during said aerobic conditions. In another embodiment, the method involves further growing under one or more additional successive aerobic -heterotrophic and anaerobic -phototrophic conditions.

In a further embodiment, the method uses all of the CO 2 , so there is no residual CO 2 released as a byproduct of the method of the invention.

Cellulase refers to a group of enzymes which, acting together hydrolyze cellulose, hemicellulose, and/or lignin. It is typically referred to as a class of enzymes produced by microorganisms (i.e., an extracellular cellulase producer), such as archaea, fungi, bacteria, protozoans, that catalyze the cellulolysis (or hydrolysis) of cellulose. However, it should be noted that there are cellulases produced by other kinds of microorganisms.

It is important to note that the present invention can utilize any microorganism strain that is an extracellular and/or intracellular cellulase, hemicellulase, and laccase enzyme producer microorganism. Such microorganism produces one or more cellulases selected from the group consisting of: endoglucanase, exoglucanase, and β-glucosidase, hemicellulases, and optionally laccase. The extracellular and/or intracellular cellulase, hemicellulase, and laccase enzyme producer is selected from the group consisting of: prokaryote, bacteria, archaea, eukaryote, yeast and fungi.

Examples of cellulase producing microorganisms that can be utilized in the present invention include those in Table 1.

Accordingly, the cellulase enzymes produced by the microorganism can perform enzymatic hydrolysis on the mixture in step (ii). At the end of the enzymatic hydrolysis, the resultant medium can contain glucose, cellobiose, acetic acid, furfural, lignin, xylose, arabinose, rhamnose, mannose, galactose, and/or other hemicelluloses sugars. Again, the present invention can utilize any microorganism that is an extracellular and/or intracellular cellulase enzyme producer to produce the requisite cellulase enzymes for enzymatic hydrolysis in step (ii) and (iv). As such, any prokaryote, including bacteria, archaea, and eukaryote, including fungi, which produces extracellular and/or intracellular cellulase enzymes may be utilized as the microorganism strain.

In one embodiment, the extracellular and/or intracellular cellulase producer is a fungus, archaea or bacteria of a genus selected from the group consisting of Humicola, Trichoderma, Penicillium, Ruminococcus, Bacillus, Cytophaga, Sporocytophaga, Humicola grisea, Trichoderma harzianum, Trichoderma lignorum, Trichoderma reesei, Penicillium verruculosum, Ruminococcus albus, Bacillus subtilis, Bacillus thermoglucosidasius, Cytophaga spp., Sporocytophaga spp., Clostridium lentocellum and Fusarium oxysporum.

In addition, a microorganism that is an extracellular and/or intracellular laccase enzyme producer may also be utilized in the present invention. Accordingly, any prokaryote, including bacteria, archaea, and eukaryote, including fungi, which produces extracellular and/or intracellular laccase may be utilized as the microorganism strain. In one embodiment, the extracellular and/or intracellular laccase producer is a fungus, bacteria or archaea of a genus selected from the group consisting of Humicola, Trichoderma, Penicillium, Ruminococcus, Bacillus, Cytophaga and Sporocytophaga. According to still a further embodiment the extracellular and/or intracellular laccase producer can be at least microorganism selected from the group consisting of Humicola grisea, Trichoderma harzianum, Trichoderma lignorum, Trichoderma reesei, Penicillium verruculosum, Ruminococcus albus, Bacillus subtilis, Bacillus thermoglucosidasius, Cytophaga spp., Sporocytophaga spp., Clostridium lentocellum and Fusarium oxysporum.

Examples of laccase producing microorganisms that can be utilized in the present invention include those in Table 2.

In one embodiment, the microorganism strain is a bacterium, such as Fusarium oxysporum.

Again, any microorganism that is an extracellular and/or intracellular cellulase enzyme producer or laccase enzyme producer can be utilized in the present to produce the requisite enzymes for the method. Examples include those listed in Tables 1 and 2.

In the present invention, the type of microorganism can be selected and/or evolved to be specific to the type of plant biomass used.

Such microorganism hydrolyzes cellulose, hemicellulose, xylose, mannose, galactose, rhamnose, arabinose or other hemicullulose sugars in the mixture. Such microorganism metabolizes cellulose and thereby produces at least one fermentation product selected from the group consisting of: Acetate, Acetone, 2,3- Butanediol, Butanol, Butyrate, CO2, Ethanol, Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, Succinate,, and other fermentation products.

The microorganism strain is tolerant to one or more compounds produced by the biomass pretreatment procedure, such as acid or alkaline pretreatment. Such compounds produced in the biomass pretreatment step can include, for example, furfural, 3,4- dihydroxybenzaldehyde, 3-methoxy-4-hydroxy-benzoic acid, cinnamic acid, vanillin, vanillin alcohol, acetic acid, lignin and other residual salts or impurities.

In a preferred embodiment, the method of present invention utilizes at least one microorganism that has been evolutionarily modified and specialized for the specific type of biomass used. The evolutionarily modified microorganism can metabolize (enzymatic hydrolysis) the pretreated targeted biomass more efficiently and such microorganisms can be better able to tolerate residual compounds, for example, furfural and acetic acid. In this respect, the evolutionarily modified microorganism has greater tolerance to furfural and acetic acid as compared to the unmodified wild-type version of the microorganism.

The evolutionarily modified microorganism can also produce one or more cellulase and/or laccase enzymes that are less inhibited by lignin and/or have improved capacity to metabolize lignin. As such, the evolutionarily modified microorganism can have improved capacity to produce enzymes (such as laccase) that metabolize lignin. Thus, the cellulase, hemicellulase and/or laccase enzymes produced by the evolutionarily modified microorganism can have greater capacity to metabolize cellulose and hemicelluloses with lignin as compared to the unmodified wild-type version of the microorganism.

Due to the use of the evolutionarily modified microorganism, the present invention allows for production of cellulases in situ in the mixture/medium. Consequently, there is no need to buy expensive cellulase enzymes from outside suppliers. This reduces operational costs as compared to conventional methods for biofuel production. Further, also due to the use of the evolutionarily modified microorganism, there is no need to wash and detoxify the acid or alkaline pretreated mixture in the present invention to remove furfural, acetic acid, and salts that would normally inhibit biofuel production (as in conventional methods). By removing the wash and detoxification steps, the present invention can further reduce operational costs as compared to conventional methods for biofuel production.

It is noted that an evolutionarily modified microorganism is defined as a microorganism that has been modified by natural selection techniques. These techniques include, for example, serial transfer, serial dilution, Genetic Engine, continuous culture, and chemostat. One method and chemostatic device (the Genetic Engine; which can avoid dilution resistance in continuous culture) has been described in U.S. Patent No. 6,686,194-Bl, incorporated herein by reference.

In one embodiment, the microorganism is evolutionarily modified by use of the continuous culture procedure as disclosed in PCT Application No. PCT/US05/05616, or United States Patent Application No. 11/508,286, each incorporated herein by reference.

By cultivating a microorganism in this manner, beneficial mutations will occur to produce brand new alleles (i.e., variants of genes) that improve an organism's chances of survival and/or growth rate in that particular environment.

As such, the microorganism (e.g., fungi, archaea, algae, or bacteria) of the present invention can constitute a different strain, which can be identified by the mutations acquired during the course of culture, and these mutations, may allow the new cells to be distinguished from their ancestors' genotype characteristics. Thus, one can select new strains of microorganisms by segregating individuals with improved rates of reproduction through the process of natural selection.

Selection parameters for evolutionarily modifying the microorganism. By way of example, the microorganism in step (i) can be evolutionarily modified, through a natural selection technique, so that through evolution, it evolves to be adapted to use the particular carbon source selected. This involves identifying and selecting the fastest growing variant microorganisms, through adaptation in the natural selection technique utilized (such as continuous culture), that grow faster than wild-type on a particular carbon source. This also includes selecting those variant microorganisms that have improved tolerance to furfural, to acetic acid or to any residual compound when using dilute acid or alkaline pre-treatment; or selecting variant microorganisms that produce one or more cellulase and/or laccase enzymes that are less inhibited by lignin and/or have improved capacity to metabolize lignin. This would also involve selecting those producing the above-discussed requisite cellulose enzymes.

It should be noted that, by using such parameters, any one of the natural selection techniques could be used in the present invention to evolutionarily modify the microorganism in the present invention.

Accordingly, the microorganisms can be evolutionarily modified in a number of ways so that their growth rate, viability, and utility as a biofuel, or other hydrocarbon product can be improved. Thus, the microorganisms can be evolutionarily modified to enhance their ability to grow on a particular substrate, constituted of the biomass and residual chemical related to chemical pre-treatment if any. In this regard, the microorganisms can be evolutionarily modified for a specific biomass plant and eventually associated residual chemicals.

The microorganisms (e.g., fungi, algae or bacteria) are preferably naturally occurring and have not been modified by recombinant DNA techniques. In other words, it is not necessary to genetically modify the microorganism to obtain a desired trait. Rather, the desired trait can be obtained by evolutionarily modifying the microorganism using the techniques discussed above. Nonetheless, even genetically modified microorganisms can be evolutionarily modified to increase their growth rate and/or viability by recombinant DNA techniques.

In one embodiment of the invention, the microorganism is anaerobic and aerobic fungus or bacterium, and in particular, Fusarium oxysporum that has been evolutionarily modified by continuous culture.

In the invention, cellulase activity and/or the amount of fermentation products can be measured using common techniques, to determine the cellulase activity and quantity of the fermentation product in the supernatant, before proceeding to the next step.

It should be noted that, in step (iii), i.e., growth under anaerobic conditions, the inoculated microorganism strain catalyzes the cellulose into fermentation products (secondary metabolites). The fermentation products comprise one or more alcohols, also CO 2 when in phototrophic condition, and soluble sugars as xylose, arabinose, rhamnose, mannose, galactose, and other hemicelluloses sugars that can then be used by the algae in step (iv). In step (iii) under anaerobic -heterotrophic conditions, the at least one algae strain uses part of said glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by the microorganism. And when step (iii) is run in anaerobic - phototrophic condition the at least one algae strain can use the released CO 2 and part or all of the fermentation products and part of said glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced by the microorganism.

Such fermentation products can include Acetate, Acetone, 2,3-Butanediol, Butanol, Butyrate, CO2, Ethanol, Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, Succinate, and such released sugars can include glucose, cellobiose, xylose, mannose, arabinose, rhamnose, galactose and/or other hemicellulose sugars.

After growing under the anaerobic conditions of step (iii), whether heterotrophic or phototrophic, the mixture is grown under further an aerobic -heterotrophic condition in step (iv). Under this additional aerobic -heterotrophic condition, the algae strain metabolizes the fermentation product produced in step (iii) to produce one or more fatty acids. Also, in step (iv), the microorganism strain continues to produce one or more cellulases, hemicellulases, and/or laccases.

Step (v) involves an optional recovery step to recover the fatty acids produced by the algae in step (iv).

Phototrophic and/or heterotrophic algae can be used in aerobic and/or anerobic environmental conditions. Such algae can use at least one of Acetate, Acetone, 2,3- Butanediol, Butyrate, CO2, Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, Succinate, and at least one of glucose, cellobiose, xylose, arabinose, rhamnose, galactose, mannose and other hemicellulose sugars under conditions so that said algae strain produces one or more fatty acids.

The growth of said at least one algae strain is not inhibited by the presence of one or more of lignin, furfural, salts and cellulases enzymes present in the mixture.

The algae strain can also grow in one or more of the conditions selected from the group consisting of aerobic, anaerobic, phototrophic, and heterotrophic conditions.

Similar to the microorganism, the algae may be evolutionarily modified (using the natural selection techniques discussed above) to serve as an improved source of fatty acids, biofuel, biodiesel, and other hydrocarbon products. In this regard, the algae can be cultivated for use as a biofuel, biodiesel, or hydrocarbon based product.

Most algae need some amount of sunlight, carbon dioxide, and water. As a result, algae are often cultivated in open ponds and lakes. However, when algae are grown in such an "open" system, the systems are vulnerable to contamination by other algae and bacteria.

In one embodiment, the present invention can utilize heterotrophic algae (Stanier et al, Microbial World, Fifth Edition, Prentice-Hall, Englewood Cliffs, New Jersey, 1986, incorporated herein by reference), which can be grown in a closed reactor.

While a variety of algal species can be used, algae that naturally contain a high amount of lipids, for example, about 15-90%, about 30-80%, about 40-60%, or about 25- 60% of lipids by dry weight of the algae is preferred. Prior to the work of the present invention, algae that naturally contained a high amount of lipids and high amount of bio- hydrocarbon were associated as having a slow growth rate. Evolutionarily modified algae strains can be produced in accordance with the present invention that exhibit an improved growth rate.

The conditions for growing the algae can be used to modify the algae. For example, there is considerable evidence that lipid accumulation takes place in algae as a response to the exhaustion of the nitrogen supply in the medium. Studies have analyzed samples where nitrogen has been removed from the culture medium and observed that while protein contents decrease under such conditions, the carbohydrate content increases, which are then followed by an increase in the lipid content of the algae. (Richardson et al, EFFECTS OF NITROGEN LIMITATION ON THE GROWTH OF ALGAE ON THE GROWTH AND COMPOSITION OF A UNICELLULAR ALGAE IN CONTINUOUS CULTURE CONDITIONS, Applied Microbiology, 1969, volume 18, page 2245-2250, 1969, incorporated herein by reference).

The algae can be evolutionarily modified by a number of techniques, including, for example, serial transfer, serial dilution, genetic engine, continuous culture, and chemostat. Any one of these techniques can be used to modify the algae. In one embodiment, the algae can be evolutionarily modified by continuous culture, as disclosed in PCT Application No. PCT/US05/05616, or United States Patent Application No. 11/508,286, each incorporated herein by reference.

In doing so, the microorganisms and the algae can be evolutionarily modified in a number of ways so that their growth rate, viability, and utility as a biofuel, or other hydrocarbon product can be improved. Accordingly, the microorganisms and algae can be evolutionarily modified to enhance their ability to grow on a particular substrate.

Selection parameters for evolutionarily modifying the algae. By way of example, the algae in step (iii) can be evolutionarily modified, through a natural selection technique, such as continuous culture, so that through evolution, the algae evolve to be adapted to use the particular carbon source selected. This involves identifying and selecting the fastest growing variant algae, through adaptation in the natural selection technique utilized, that grow faster than wild-type on a particular carbon source. This also includes, for example, selecting those algae that use acetic acid as a carbon source with improved tolerance to lignin, furfural and salts. It should be noted that, by using such parameters, any one of the natural selection techniques could be used in the present invention to evolutionarily modify the algae in the present invention.

In the present invention, such evolutionarily modified algae metabolize one or more compounds selected from the group consisting of: glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars and/or waste glycerol, and the algae use one or more of the fermentation products as Acetate, Acetone, 2,3-Butanediol, Butanol, Butyrate, CO2, Ethanol, Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, Succinate, as a carbon source, under conditions so that said at least one algae strain produces one or more fatty acids. Such evolutionarily modified algae can also grow in one or more of the conditions selected from the group consisting of aerobic, anaerobic, phototrophic, and heterotrophic conditions. In one embodiment, when the invention is performed under aerobic and heterotrophic conditions, the algae use respiration.

In step (iv), the algae using the same amount of carbon source as an organism producing fermentation by-product producer, will produce only up to 10% carbon dioxide. In this regard, more sugar is used by the algae for growth than is transformed to carbon dioxide. Alternatively, the microorganism or algae can be one that does not use fermentation, and as such much less carbon dioxide is made as a by-product in respiration.

Also, said at least one algae strain produces no inhibitory by-product, for growth of said algae. The growth of said algae is not inhibited by the presence of one or more of lignin, furfural, salts, cellulase enzymes and hemicellulase enzymes.

Types of algae that can be utilized in the invention is one or more selected from the group consisting of green algae, red algae, blue-green algae, cyanobacteria and diatoms.

It should be noted that the present invention can utilize any algae strain that metabolizes said at least one fermentation products, including acetic acid, ethanol, glucose, cellobiose, xylose or other hemicellulose sugars, pyruvate and succinate, under conditions so that said algae strain produces one or more fatty acids.

By way of example, the algae utilized in step (iii) can be from the following taxonomic divisions of algae:

(1) Division Chlorophyta (green algae);

(2) Division Cyanophyta (blue-green algae);

(3) Division Bacillariophyta (diatoms);

(4) Division Chrysophyta;

(5) Division Xanthophyta;

(6) Division Cryptophyta;

(7) Division Euglenophyta;

(8) Division Ochrophyta ;

(9) Division Haptophyta; and

(10) Division Dinophyta.

More specifically, the algae can be from the following species of algae, included within the above divisions (wherein number in parenthesis corresponds to the division): Biddulphia (8); Pinguiococcus (8); Skeletonema (8); Emiliania (9); Prymnesium (9); Crypthecodinium (10); Anabaenopsis circularis (2); Ankistrodesmus braunii (1); A.falcatus (1); Botrydiopsis intercedens (5); Bracteacoccus cinnabarinus (1); B. engadiensis (1); B. minor (Chodat) Petrova (1);

B. terrestris (1); Bracteacoccus sp. (1); Bracteacoccus sp. (1); Bumilleriopsis brevis (5); Chilomonas Paramecium (6); Chlamydobotrys sp. (1); Chlamydomonas agloeformis (1);

C. dysosmos (1);

C. mundana Mojave strain Boron strain (1);

C. reinhardi (-) strain (1);

Chlorella ellipsoidea (1);

C. protothecoides (1);

C. pyrenoidosa (1);

C. pyrenoidosa ATCC 7516 (1);

C pyrenoidosa C-37-2 (1);

C pyrenoidosa Emerson (1);

C pyrenoidosa 7-11-05 (1);

C vulgaris (1);

C vulgaris ATCC 9765 (1);

C vulgaris Emerson (1);

C vulgaris Pratt-Trealease (1);

C vulgaris var. viridis (1);

Chlorellidium tetrabotrys (5);

Chlorocloster engadinensis (5);

Chlorococcum macrostigmatum (1); Chlorococcum sp. (1);

Chlorogloea fritschii (2);

Chlorogonium elongatum (1);

Coccomyxa elongata (1);

Cyclotella sp. (3);

Dictyochloris fragrans (1);

Euglena gracilis (7);

E. gracilis Vischer (7);

E. gracilis var. bacillaris (7);

E. gracilis var. saccharophila (7);

Haematococcus pluvialis (1);

Navicula incerta Grun. (3);

N. pelliculosa (3);

Neochloris alveolaris (1);

N. aquatica Starr (1);

N. gelatinosa Herndon (1);

N. pseudoalveolaris Deason (1);

Neochloris sp. (1);

Nitzschia angularis var. afflnis (3) (Grun.) perag. ,

N. chlosterium (Ehr.) (3);

N. curvilineata Hust. (3);

N. filiformis β);

N. frustulum (Kurtz.) (3);

N. laevis Hust. (3);

Nostoc muscorum (2);

Ochromonas malhamensis (4);

Pediastrum boryanum (1);

P. duplex (1);

Polytoma obtusum (1);

P. ocellatum (1);

P. uvella (1);

Polytomella caeca (or coeca) (1);

Prototheca zopfli (1);

Scenedesmus acuminatus (1); S. acutiformis (1);

S. costulatus Chod, var. chlorelloides (1);

S. dimorphus (1);

S. obliquus (1);

S. quadricauda (1);

Spongiochloris excentrica (1);

S. lamellata Deason (1);

S. spongiosus (1);

Spongiochloris sp. (1);

Spongiococcum alabamense (1);

S. excentricum (1);

S. excentricum Deason etBold (1)

S. multinucleatum (1);

Stichococcus bacillaris (1);

S. subtilis (1);

Tolypothrix tenuis (2);

Tribonema aequale (5); and

T. minus (5).

In one embodiment, the algae can be from Chlorophyta (Chlorella and Prototheca), Prasinophyta (Dunaliella), Bacillariophyta (Navicula and Nitzschia), Ochrophyta (Ochromonas), Dinophyta (Gyrodinium) and Euglenozoa (Euglena). More preferably, the algae is one selected from the group consisting of: Monalanthus Salina; Botryococcus Braunii; Chlorella prototecoides; Outirococcus sp.; Scenedesmus obliquus; Nannochloris sp.; Dunaliella bardawil (D. Salina); Navicula pelliculosa; Radiosphaera negevensis; Biddulphia aurita; Chlorella vulgaris; Nitzschia palea; Ochromonas dannica; Chrorella pyrenoidosa; Peridinium cinctum; Neochloris oleabundans; Oocystis polymorpha; Chrysochromulina spp. ; Scenedesmus acutus; Scenedesmus spp.; Chlorella minutissima; Prymnesium parvum; Navicula pelliculosa; Scenedesmus dimorphus; Scotiella sp.; Chorella spp.; Euglena gracilis; and Porphyridium cruentum.

Examples of algae that can be utilized in the present invention include those in Tables 3 and 4.

In another embodiment, the algae strain is Chlorella protothecoides and has been evolutionarily modified by continuous culture using the techniques and procedures described above. Cyanobacteria may also be used with the present invention. Cyanobacteria are prokaryotes (single-celled organisms) often referred to as "blue-green algae." While most algae is eukaryotic, cyanobacteria is the most common exception. Cyanobacteria are generally unicellular, but can be found in colonial and filamentous forms, some of which differentiate into varying roles. For purposes of the claimed invention, cyanobacteria are considered algae.

Chlorella protothecoides and Dunaliella Salina are species that have been evolutionarily modified, cultivated, and harvested for production of a biodiesel.

The following publications relate to growing different types of algae and then harvesting algae for the purpose of producing biodiesel are incorporated herein by reference:

- Xu et al, HIGH QUALITY BIODESEL PRODUCTION FROM A MICROALGA CHLORELLA PROTHECOIDES BY HETEROTROPHIC GROWTH IN FERMENTERS, Journal of Biotechnology, vol. 126, 499-507, 2006,

- Kessler, Erich, PHYSIOLOGICAL AND BIOCHEMICAL CONTRIBUTIONS TO THE TAXONOMY OF THE GENUS PROTOTHECA, III. UTILIZATION OF ORGANIC CARBON AND NITROGEN COMPOUNDS, Arch Microbiol, volume 132, 103-106, 1982,

- Johnson D, 1987, OVERVIEW OF THE DOE/SERI AQUATIC SPECIES PROGRAM FY 1986 SOLAR ENERGY INSTITUTE,

- Pratt et al, PRODUCTION OF PROTEIN AND LIPID BY CHLORELLA VULGARIS AND CHLORELLA PYRENOIDOSA, Journal of Pharmaceutical Sciences, volume 52, Issue 10, 979-984 2006, and

- Sorokin, MAXIMUM GROWTH RATES OF CHLORELLA IN STEADY-STATE AND IN SYNCHRONIZED CULTURES, Proc. N.A.S, volume 45, 1740-1743, 1959.

- J.E. Zajic and Y.S. Chiu, HETEROTROPHIC CULTURE OF ALGAE, Biochemical Engineering, Faculty of Engineering Science, University of Western Ontario, London.

By employing the methods of the instant invention, the inoculation and culture of the mixture with the at least one algae strain in step (ii) results in the algae metabolizing at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars. In step (iii), when in heterotrophic condition the algae strain uses part of the the glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced the microorganism in step (ii), and when in phototrophic condition the algae strain uses most of the released CO 2 and of the fermentation products and part of the the glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars produced the microorganism in step (ii). In step (iv), the algae metabolizes at least one of the fermentation products, which can include Acetate, Acetone, 2,3-Butanediol, Butanol, Butyrate, CO2, Ethanol, Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, Succinate, under conditions so that said at least one algae strain produces one or more compounds, including fatty acids.

The present invention involves culturing and growing the evolutionarily modified algae for extracellular and/or intracellular production of one or more compounds, such as fatty acids, hydrocarbons, proteins, pigments, sugars, such as polysaccharides and monosaccharides, and glycerol.

The resultant fatty acids, hydrocarbons, proteins, pigments, sugars, such as polysaccharides and monosaccharides, and glycerol in the algae can be used for biofuel, cosmetic, alimentary, mechanical grease, pigmentation, and medical use production.

In optional step (v), the fatty acids, hydrocarbons, proteins, pigments, sugars, such as polysaccharides and monosaccharides, and glycerol are recovered from the algae. The recovery step can be done by conventional techniques including one or more of fractionating the algae in the culture to obtain a fraction containing the compound, and other techniques including filtration-centrifugation, flocculation, solvent extraction, acid and base extraction, ultrasonication, microwave, pressing, distillation, thermal evaporation, homogenization, hydrocracking (fluid catalytic cracking), and drying of said at least one algae strain containing fatty acids.

In one embodiment, the resultant supernatant recovered in step (v) can be reused.

Moreover, the recovered fatty acids can be optionally isolated and chemically treated (e.g., by transesterification), and thereby made into a biofuel (biodiesel) that can be incorporated into an engine fuel.

In this regard, the algae strain of the present invention produces hydrocarbon chains which can be used as feedstock for hydrocracking in an oil refinery to produce one or more compounds selected from the group consisting of octane, gasoline, petrol, kerosene, diesel and other petroleum product as solvent, plastic, oil, grease and fibers.

Direct transesterification can be performed on cells of the algae strain to produce fatty acids for biodiesel fuel. Methods of direct transesterification are well known and include breaking the algae cells, releasing fatty acids and transesterification through a base or acid method with methanol or ethanol to produce biodiesel fuel.

A further advantage of the method of the present invention is that the algae strain can be adapted to use waste glycerol, as a carbon source, produced by the transesterification reaction without pretreatment or refinement to produce fatty acids for biodiesel production. Raw glycerol is the by-product of a transesterification reaction comprising glycerol and impurities such as fatty acid components, oily components, acid components, alkali components, soap components, alcohol component (e.g., methanol or ethanol) solvent (N- hexane) salts and/or diols. Due to the number and type of impurities present in raw glycerol, microorganisms exhibit little to no growth on the raw glycerol itself. However, the microorganism (e.g., algae or bacteria) can be evolutionarily modified to utilize raw glycerol as a primary carbon source.

The initial test for determining whether a particular type of microorganism will be able to grow in the presence of raw glycerol is the Refined Glycerol Test. The Refined Glycerol Test comprises culturing the microorganism in a medium comprising refined glycerol. The medium utilized in the Refined Glycerol Test may or may not have another carbon source such as glucose. However, the medium in the Refined Glycerol Test must contain a sufficient amount of glycerol so that it can be determined that the microorganism exhibits a minimum metabolizing capacity of the microorganism. The medium can contain about 10ml-50 ml per liter of refined glycerol, about O. lml-lOOml per liter of refined glycerol, or about 2ml- 15ml per liter of refined glycerol.

If a positive result (i.e., the microorganism grows in the medium) is obtained with the Refined Glycerol Test, the microorganism can be evolutionarily modified to grow in a medium comprising raw glycerol. The culture medium can comprise about 10-100% raw glycerol as a carbon source, about 20-90% raw glycerol as a carbon source, about 30-75% raw glycerol as a carbon source, about 40-75% raw glycerol as a carbon source, or about 50.01-55% raw glycerol as a carbon source. Indeed, some strains of microorganisms have been evolutionary modified to grow on a culture medium containing 100% raw glycerol.

An evolutionarily modified microorganism which produces extracellular and/or intracellular cellulase, hemicellulase, and laccase obtained in accordance with the present invention has a maximum growth rate using the specific carbon sources in the pretreated biomass mixture of at least 5%, preferably 10%, 15%, 25%, 50%, 75%, 100%, 200%, 25%- 100%, 25%-100%, 50%-150%, 25-200%, more than 200%, more than 300%, or more than 400% greater than microorganism of the same species that has not been evolutionarily modified to perform in the present invention.

An evolutionarily modified algae obtained in accordance with the present invention has a maximum growth rate using, as a carbon source, the released polysaccharide and monosaccharide sugars from step (i) in the pretreated biomass mixture of at least 5%, preferably 10%, 15%, 25%, 50%, 75%, 100%, 200%, 25%-100%, 25%-100%, 50%-150%, 25-200%, more than 200%, more than 300%, or more than 400% greater than algae of the same species that has not been evolutionarily modified to perform in the present invention.

While it is envisioned that the most important commercial use for microorganisms grown from the by-products of biodiesel production will be to use the microorganisms themselves for products such as biofuel, biodiesel, "bio"-hydrocarbon products, renewable hydrocarbon products, and fatty acid based products, the invention is not limited to this embodiment. For example, if the microorganism is an algae, the algae could be grown from the by-products of biofuel production and harvested for use as a food, medicine, and nutritional supplement.

The biofuel obtained from the present invention may be used directly or as an alternative to petroleum for certain products.

In another embodiment, the biofuel (e.g., biodiesel) of the present invention may be used in a blend with other petroleum products or petroleum alternatives to obtain fuels such as motor gasoline and distillate fuel oil composition; finished nonfuel products such as solvents and lubricating oils; and feedstock for the petrochemical industry such as naphtha and various refinery gases.

For example, the biofuel as described above may be used directly in, or blended with other petroleum based compounds to produce solvents; paints; lacquers; and printing inks; lubricating oils; grease for automobile engines and other machinery; wax used in candy making, packaging, candles, matches, and polishes; petroleum jelly; asphalt; petroleum coke; and petroleum feedstock used as chemical feedstock derived from petroleum principally for the manufacture of chemicals, synthetic rubber, and a variety of plastics.

In a preferred embodiment, biodiesel produced in accordance with the present invention may be used in a diesel engine, or may be blended with petroleum-based distillate fuel oil composition at a ratio such that the resulting petroleum substitute may be in an amount of about 5-95%, about 15-85%, about 20-80%, about 25-75%, about 35-50%, about 50-75%, or about 75-95% by weight of the total composition. The components may be mixed in any suitable manner.

The process of fueling a compression ignition internal combustion engine, comprises drawing air into a cylinder of a compression ignition internal combustion engine; compressing the air by a compression stroke of a piston in the cylinder; injecting into the compressed air, toward the end of the compression stroke, a fuel comprising the biodiesel; and igniting the fuel by heat of compression in the cylinder during operation of the compression ignition internal combustion engine. In another embodiment, the biodiesel is used as a lubricant or in a process of fueling a compression ignition internal combustion engine.

Alternatively, the biofuel may be further processed to obtain other hydrocarbons that are found in petroleum such as paraffins (e.g., methane, ethane, propane, butane, isobutane, pentane, and hexane), aromatics (e.g., benzene and naphthalene), cycloalkanes (e.g., cyclohexane and methyl cyclopentane), alkenes (e.g., ethylene, butene, and isobutene), alkynes (e.g., acetylene, and butadienes).

The resulting hydrocarbons can then in turn be used in petroleum based products such as solvents; paints; lacquers; and printing inks; lubricating oils; grease for automobile engines and other machinery; wax used in candy making, packaging, candles, matches, and polishes; petroleum jelly; asphalt; petroleum coke; and petroleum feedstock used as chemical feedstock derived from petroleum principally for the manufacture of chemicals, synthetic rubber, and a variety of plastics.

The following examples are but two embodiments of the invention. It will be apparent that various changes and modifications can be made without departing from the scope of the invention as defined in the claims. Examples

One exemplified embodiment of the method of the present invention can be found in the chart in Fig. 4 and is discussed below.

In this example (A), a plant biomass material of chipped switchgrass was subjected to pretreatment by acid hydrolysis (sulfuric acid 0.5 to 2.0%) and heat treatment (120-200 0 C). This pretreatment procedure produced a mixture for use in the above-discussed step (i). This mixture contained among other things cellulose, hemicellulose, lignin, furfural, and acetic acid.

In step (i), the mixture was inoculated with an evolutionarily modified microorganism strain of Fusarium oxysporum (designated EVG41025) and an evolutionarily modified algae strain of Chlorella protothecoides (designated EVG17020). The strains were grown under heterotrophic conditions, and under alternating aerobic and anerobic conditions. The conditions and strains are defined below.

• The modified Fusarium oxysporum strain (EVG41025) was evolved to metabolize pretreated switchgrass more efficiently as a carbon source and produces fermentation products, such as: Acetate, Acetone, 2,3-Butanediol, Butanol, Butyrate, CO2, Ethanol,

Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, Succinate, and other fermentation products. • The modified Fusarium oxysporum strain (EVG41025) was evolved to tolerate furfural and acetic acid better and the presense of lignin. The strain produces external cellulase enzymes specific for switchgrass.

• Step (ii) involved growth of Fusarium oxysporum (EVG41025) and Chlorella protothecoides (EVG 17020) in an aerobic environment.

• Under the aerobic conditions in step (ii), Fusarium oxysporum (EVG41025) produced cellulases, hemicellulases and laccases that hydrolyzed cellulose, hemicellulose and lignin and produced glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulse sugars that were metabolized by Chlorella protothecoides (EVG 17020) that also metabolized acetic acid from the pretreatment.

• Step (iii) involved growth under anaerobic conditions. Fusarium oxysporum (EVG41025) produced one or more fermentation products and Chlorella protothecoides (EVG 17020) used part of the sugars produced by Fusarium oxysporum (EVG41025).

• Step (iv) involved growing under aerobic conditions. Chlorella protothecoides (EVG 17020) metabolized the fermentation products produced in step (iii) to produce fatty acids, and Fusarium oxysporum (EVG41025) continues to produce cellulases.

• Chlorella protothecoides (EVG 17020) was evolved to heterotrophically use as carbon sources the fermentation products released by EVG41025 and any soluble sugars released by the enzymatic activity of EVG41025.

• Chlorella Protothecoides (EVG 17020) metabolizes: acetic acid, ethanol, and other fermentation products like succinate, butyrate, pyruvate, waste glycerol, and it uses acetic acid as a carbon source, and any soluble sugars released by the pretreatment and fermentation of switchgrass.

• Presence of lignin, furfural and salts do not inhibit growth.

• Chlorella Protothecoides (EVG 17020) produces 40% or more fatty acid (cell dry weight).

In the method, the microorganism and the algae were grown under heterotrophic conditions and the algae produced fatty acids.

In step (v), the algae cells and fatty acids were then recovered by filtration and cell drying.

Direct transesterification was then performed on the dry cells (ultrasonication, membrane rupture, through a base or acid method with methanol or ethanol) to produce biodiesel fuel. Waste glycerol was also recovered and recycled. The resultant biodiesel fuel was then directly used in any diesel engine for cars, trucks, generators, boats, etc.

Another exemplified embodiment of the method of the present invention can be found in the chart in Fig. 5 and is discussed below.

In this example (B), a plant biomass material of chipped switchgrass was subjected to pretreatment by acid hydrolysis (sulfuric acid 0.5 to 2.0%) and heat treatment (120-200 0 C). This pretreatment procedure produced a mixture for use in the above-discussed step (i). This mixture contained among other things cellulose, hemicellulose, lignin, furfural, and acetic acid.

In step (i), the mixture was inoculated with an evolutionarily modified microorganism strain of Fusarium oxysporum (designated EVG42050) and an evolutionarily modified algae strain of Chlorella protothecoides (designated EVG17075). In steps (ii)-(iv), the strains were grown under aerobic-heterotrophic conditions (step (U)), and then anaerobic-phototrophic conditions (step (Ui)) and then under aerobic-heterotrophic conditions (step (iv)). The conditions and strains are defined below.

• The modified Fusarium oxysporum strain (EVG42050) was evolved to metabolize pretreated switchgrass more efficiently as a carbon source and produces fermentation products, such as: Acetate, Acetone, 2,3-Butanediol, Butanol, Butyrate, CO 2 , Ethanol, Formate, Glycolate, Lactate, Malate, Propionate, Pyruvate, Succinate, and other fermentation products.

• The modified Fusarium oxysporum strain (EVG42050) was evolved to tolerate furfural and acetic acid better and the presense of lignin. The strain produces external cellulase enzymes specific for switchgrass.

• Step (U) involved growth of Fusarium oxysporum (EVG42050) and Chlorella protothecoides (EVG 17075) in an aerobic-heterotrophic environment.

• Under aerobic-heterotrophic conditions in step (U), Fusarium oxysporum (EVG42050) produced cellulases, hemicellulases and laccases that hydrolyzed cellulose, hemicellulose and lignin and produced glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulse sugars that were then metabolized by Chlorella protothecoides (EVG 17075) that also metabolized acetic acid from the pretreatment.

• Step (Ui) involved growth under anaerobic-phototrophic conditions. Fusarium oxysporum (EVG42050) produced one or more fermentation products and CO 2 , and Chlorella protothecoides (EVG 17075) used most of the CO 2 , metabolized part or all of the fermentation products and used part of the sugars produced by Fusarium oxysporum (EVG42050).

• Step (iv) involved growing under aerobic -heterotrophic conditions. Chlorella protothecoides (EVG 17075) metabolized the fermentation products produced in step (iii) to produce fatty acids, and Fusarium oxysporum (EVG42050) continues to produce cellulases.

• Chlorella protothecoides (EVG 17075) was evolved to heterotrophically use as carbon sources the fermentation products released by EVG42050 and any soluble sugars released by the enzymatic activity of EVG42050.

• Chlorella Protothecoides (EVG 17075) metabolizes: acetic acid, ethanol, and other fermentation products like succinate, butyrate, pyruvate, waste glycerol, and it uses acetic acid as a carbon source, and any soluble sugars released by the pretreatment and fermentation of switchgrass.

• Presence of lignin, furfural and salts do not inhibit growth.

• Chlorella Protothecoides (EVG 17075) produces 40% or more fatty acid (cell dry weight).

In the method, the microorganism and the algae were alternatively grown under heterotrophic and phototrophic conditions and the algae produced fatty acids.

In step (v), the algae cells and fatty acids were then recovered by filtration and cell drying.

Direct transesterification was then performed on the dry cells (ultrasonication, membrane rupture, through a base or acid method with methanol or ethanol) to produce biodiesel fuel. Waste glycerol was also recovered and recycled. The resultant biodiesel fuel was then directly used in any diesel engine for cars, trucks, generators, boats, etc. The method used most of the released CO 2 , so there is little residual CO 2 released as a byproduct of said method.

While the invention has been described and pointed out in detail with reference to operative embodiments thereof it will be understood by those skilled in the art that various changes, modifications, substitutions and omissions can be made without departing from the spirit of the invention. It is intended, therefore, that the invention embrace those equivalents within the scope of the claims which follow. TABLE 1 - EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA- AND/OR INTRA-CELLULAR CELLULASE ENZYMES

TABLE 2 - EXAMPLES OF MICRO-ORGANISMS PRODUCING EXTRA- AND/OR INTRA-CELLULAR LACCASE ENZYMES

TABLE 3 - EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA- AND/OR INTRA-CELLULAR CELLULASE ENZYMES

ALGAE STRAINS

Bacillariophyta Cymbella microcephala Bacillariophyta Cymbella norvegica Bacillariophyta Cymbella pusilla Bacillariophyta Cymbella tumida Bacillariophyta Denticula kuetzingii Bacillariophyta Diadesmis confervacea Bacillariophyta Diatoma tenue var. elongatum Bacillariophyta Diploneis subovalis Bacillariophyta Encyonema minutum var. pseudogracilis Bacillariophyta Entomoneis paludosa Bacillariophyta Eucocconeis sp. Bacillariophyta Eunotia curvata Bacillariophyta Eunotia flexulosa Bacillariophyta Eunotia formica Bacillariophyta Eunotia glacialis Bacillariophyta Eunotia maior Bacillariophyta Eunotia naegelii Bacillariophyta Eunotia pectinalis Bacillariophyta Eunotia sp. Bacillariophyta Fallacia monoculata Bacillariophyta Fallacia pygmaea Bacillariophyta Fragilaria capucina Bacillariophyta Fragilaria crotonensis Bacillariophyta Fragilariforma virescens Bacillariophyta Gomphonema affine Bacillariophyta Gomphonema affine var. insigne Bacillariophyta Gomphonema angustatum Bacillariophyta Gomphonema brebissonii Bacillariophyta Gomphonema carolinense Bacillariophyta Gomphonema dichotomum Bacillariophyta Gomphonema gracile Bacillariophyta Gomphonema intracatum Bacillariophyta Gomphonema intracatum var. vibrio Bacillariophyta Gomphonema parvulum Bacillariophyta Gomphonema subclavatum var. commutatum

Bacillariophyta Gomphonema subclavatum var. mexicanum Bacillariophyta Gomphonema subtile Bacillariophyta Gomphonema truncatum Bacillariophyta Gyrosigma acuminatum Bacillariophyta Gyrosigma obtusatum Bacillariophyta Gyrosigma spencerii var. curvula Bacillariophyta Hantzschia amphioxys Bacillariophyta Hantzschia amphioxys f. capitata Bacillariophyta Hantzschia amphioxys var. maior Bacillariophyta Hantzschia elongata Bacillariophyta Hantzschia sigma Bacillariophyta Hantzschia spectabilis Bacillariophyta Hantzschia virgata var. gracilis Bacillariophyta Lemnicola hungarica Bacillariophyta Minutocellis sp. Bacillariophyta Navicula abiskoensis Bacillariophyta Navicula angusta Bacillariophyta Navicula arvensis Bacillariophyta Navicula capitata Bacillariophyta Navicula cincta Bacillariophyta Navicula cryptocephala Bacillariophyta Navicula cryptocephala var. veneta Bacillariophyta Navicula decussis Bacillariophyta Navicula erifuga Bacillariophyta Navicula gerloffii Bacillariophyta Navicula incerta Bacillariophyta Navicula libonensis Bacillariophyta Navicula menisculus var. upsaliensis Bacillariophyta Navicula minima Bacillariophyta Navicula minima var. atomoides Bacillariophyta Navicula phyllepta Bacillariophyta Navicula radiosa Bacillariophyta Navicula radiosa f. tenella Bacillariophyta Navicula radiosa var. tenella Bacillariophyta Navicula recens Bacillariophyta Navicula reinhardtii Bacillariophyta Navicula rhynchocephala var. amphiceros Bacillariophyta Navicula salinarum Bacillariophyta Navicula secura Bacillariophyta Navicula seminuloides Bacillariophyta Navicula seminulum Bacillariophyta Navicula subrhynchocephala Bacillariophyta Navicula tantula Bacillariophyta Navicula tenelloides Bacillariophyta Navicula tripunctata Bacillariophyta Navicula tripunctata var. schizonemoides Bacillariophyta Navicula trivialis Bacillariophyta Navicula viridula var. rostellata Bacillariophyta Neidium affine Bacillariophyta Neidium affine var. humerus Bacillariophyta Neidium affine var. longiceps Bacillariophyta Neidium affine var. undulatum Bacillariophyta Neidium affine var. undulatum Bacillariophyta Neidium bisulcatum Bacillariophyta Neidium bisulcatum var. subampilatum Bacillariophyta Neidium productum Bacillariophyta Nitzschia acicularis Bacillariophyta Nitzschia amphibia Bacillariophyta Nitzschia amphibioides Bacillariophyta Nitzschia communis Bacillariophyta Nitzschia commutata Bacillariophyta Nitzschia dissipata Bacillariophyta Nitzschia gracilis Bacillariophyta Nitzschia linearis Bacillariophyta Nitzschia linearis var. tenuis Bacillariophyta Nitzschia nana Bacillariophyta Nitzschia ovalis Bacillariophyta Nitzschia paleacea Bacillariophyta Nitzschia perminuta Bacillariophyta Nitzschia reversa Bacillariophyta Nitzschia rostellata Bacillariophyta Nitzschia sigma Bacillariophyta Nitzschia sp. Bacillariophyta Nitzschia subtilioides Bacillariophyta Nitzschia terricola Bacillariophyta Nitzschia vermicularis Bacillariophyta Nitzschia vitrea Bacillariophyta Orthoseira dendroteres Bacillariophyta Phaeodactylum tricornutum Bacillariophyta Pinnularia appendiculata Bacillariophyta Pinnularia biceps Bacillariophyta Pinnularia borealis Bacillariophyta Pinnularia brebissonii Bacillariophyta Pinnularia gibba Bacillariophyta Pinnularia mayeri Bacillariophyta Pinnularia mesolepta Bacillariophyta Pinnularia nodosa Bacillariophyta Pinnularia sp. Bacillariophyta Pinnularia subcapitata Bacillariophyta Pinnularia subcapitata var. Elongata Bacillariophyta Pinnularia subgibba Bacillariophyta Pinnularia termitina Bacillariophyta Pinnularia viridiformis Bacillariophyta Placoneis dementis Bacillariophyta Placoneis elginensis Bacillariophyta Pleurosigma elongatum Bacillariophyta Pleurosira laevis Bacillariophyta Pseudostaurosira construens Bacillariophyta Rhopalodia contorta Bacillariophyta Rhopalodia gibba Bacillariophyta Scoliopleura peisonis Bacillariophyta Sellaphora pupula Bacillariophyta Sellaphora pupula var. rectangularis Bacillariophyta Skeletonema costatum Bacillariophyta Stauroneis acuta Bacillariophyta Stauroneis anceps Bacillariophyta Stauroneis anceps f. gracilis Bacillariophyta Stauroneis anceps var. gracilis Bacillariophyta Stauroneis phoenicenteron Bacillariophyta Stauroneis phoenicenteron f. gracilis

Bacillariophyta Stauroneis smithii var. incisa

Bacillariophyta Staurosira construens

Bacillariophyta Staurosirella pinnata

Bacillariophyta Stenopterobia curvula

Bacillariophyta Stephanodiscus minutulus

Bacillariophyta Stephanodiscus parvus

Bacillariophyta Surirella angusta

Bacillariophyta Surirella brightwellii

Bacillariophyta Surirella cf. crumena

Bacillariophyta Surirella ovalis

Bacillariophyta Surirella ovata

Bacillariophyta Surirella ovata var. apiculata

Bacillariophyta Surirella peisonis

Bacillariophyta Surirella striatula

Bacillariophyta Synedra famelica

Bacillariophyta Synedra radians

Bacillariophyta Synedra rumpens

Bacillariophyta Synedra ulna

Bacillariophyta Synedra ulna var. chaseana

Bacillariophyta Tabellaria flocculosa

Bacillariophyta Thalassiosira pseudonana

Bacillariophyta Thalassiosira sp.

Bacillariophyta Tryblionella apiculata

Bacillariophyta Tryblionella debilis

Bacillariophyta Tryblionella gracilis

Bacillariophyta Tryblionella hungarica

Bacillariophyta Tryblionella levidensis

Cercozoa Chlorarachnion globosum

Cercozoa Chlorarachnion reptans

Chlorophyta Acetabularia acetabulum

Chlorophyta Acetabularia caliculus

Chlorophyta Acetabularia crenulata

Chlorophyta Acetabularia dentata

Chlorophyta Acetabularia farlowii

Chlorophyta Acetabularia kilneri

Chlorophyta Acetabularia major

Chlorophyta Acetabularia ryukyuensis

Chlorophyta Acicularia schenckii

Chlorophyta Actinotaenium habeebense

Chlorophyta Anadyomene stellata

Chlorophyta Ankistrodesmus angustus

Chlorophyta Ankistrodesmus arcuatus

Chlorophyta Ankistrodesmus densus

Chlorophyta Ankistrodesmus falcatus var. acicularis

Chlorophyta Ankistrodesmus falcatus var. stipitatus

Chlorophyta Ankistrodesmus nannoselene

Chlorophyta Ankistrodesmus pseudobraunii Chlorophyta Ankistrodesmus sp. Chlorophyta Aphanochaete confervicola Chlorophyta Aphanochaete confervicola var. major Chlorophyta Aphanochaete elegans Chlorophyta Aphanochaete elegans var. minor Chlorophyta Arthrodesmus sp. Chlorophyta Ascochloris multinucleata Chlorophyta Asterococcus superbus Chlorophyta Astrephomene gubernaculifera Chlorophyta Atractomorpha echinata Chlorophyta Atractomorpha porcata Chlorophyta Axilococcus clingmanii Chlorophyta Axilosphaera vegetata Chlorophyta Basicladia sp. Chlorophyta Batophora occidentalis Chlorophyta Blastophysa rhizopus Chlorophyta Boergesenia forbesii Chlorophyta Boodlea composita Chlorophyta Boodlea montagnei Chlorophyta Bornetella oligospora Chlorophyta Bornetella sphaerica Chlorophyta Borodinellopsis texensis Chlorophyta Brachiomonas submarina Chlorophyta Brachiomonas submarina var. pulsifera Chlorophyta Bracteacoccus aerius Chlorophyta Bracteacoccus cohaerans Chlorophyta Bracteacoccus giganteus Chlorophyta Bracteacoccus grandis Chlorophyta Bracteacoccus medionucleatus Chlorophyta Bracteacoccus minor var. desertorum Chlorophyta Bracteacoccus minor var. glacialis Chlorophyta Bracteacoccus pseudominor Chlorophyta Bulbochaete hiloensis Chlorophyta Bulbochaete sp. Chlorophyta Capsosiphon fulvescens Chlorophyta Carteria crucifera Chlorophyta Carteria eugametos var. contaminans Chlorophyta Carteria olivieri Chlorophyta Carteria radiosa Chlorophyta Carteria sp. Chlorophyta Centrosphaera sp. Chlorophyta Cephaleuros parasiticus Chlorophyta Cephaleuros virescens Chlorophyta Chaetomorpha auricoma Chlorophyta Chaetomorpha spiralis Chlorophyta Chaetopeltis sp. Chlorophyta Chaetophora incrassata Chlorophyta Chaetosphaeridium globosum Chlorophyta Chalmasia antillana Chlorophyta Chamaetrichon capsulatum Chlorophyta Characiochloris acuminata Chlorophyta Characiosiphon rivularis Chlorophyta Characium acuminatum Chlorophyta Characium bulgariense Chlorophyta Characium californicum Chlorophyta Characium fusiforme Chlorophyta Characium hindakii Chlorophyta Characium oviforme Chlorophyta Characium perforatum Chlorophyta Characium polymorphum Chlorophyta Characium saccatum Chlorophyta Characium typicum Chlorophyta Chlamydomonas allensworthii Chlorophyta Chlamydomonas applanata Chlorophyta Chlamydomonas asymmetrica Chlorophyta Chlamydomonas callosa Chlorophyta Chlamydomonas chlamydogama Chlorophyta Chlamydomonas cribrum Chlorophyta Chlamydomonas culleus Chlorophyta Chlamydomonas debaryana var. cristata Chlorophyta Chlamydomonas desmidii Chlorophyta Chlamydomonas euryale Chlorophyta Chlamydomonas eustigma Chlorophyta Chlamydomonas fimbriata Chlorophyta Chlamydomonas gerloffii Chlorophyta Chlamydomonas gigantea

Chlorophyta Chlamydomonas gloeophila var. irregularis Chlorophyta Chlamydomonas gyrus Chlorophyta Chlamydomonas hedleyi Chlorophyta Chlamydomonas hydra Chlorophyta Chlamydomonas inflexa Chlorophyta Chlamydomonas isabeliensis Chlorophyta Chlamydomonas leiostraca Chlorophyta Chlamydomonas lunata Chlorophyta Chlamydomonas melanospora Chlorophyta Chlamydomonas mexicana Chlorophyta Chlamydomonas minuta Chlorophyta Chlamydomonas minutissima Chlorophyta Chlamydomonas monadina Chlorophyta Chlamydomonas monoica Chlorophyta Chlamydomonas mutabilis Chlorophyta Chlamydomonas noctigama Chlorophyta Chlamydomonas oblonga Chlorophyta Chlamydomonas orbicularis Chlorophyta Chlamydomonas oviformis Chlorophyta Chlamydomonas perpusillus Chlorophyta Chlamydomonas philotes Chlorophyta Chlamydomonas proteus Chlorophyta Chlamydomonas provasolii Chlorophyta Chlamydomonas pseudagloe Chlorophyta Chlamydomonas pseudococcum Chlorophyta Chlamydomonas Pulsatilla Chlorophyta Chlamydomonas pulvinata Chlorophyta Chlamydomonas pygmaea Chlorophyta Chlamydomonas radiata Chlorophyta Chlamydomonas rapa Chlorophyta Chlamydomonas sajao Chlorophyta Chlamydomonas simplex Chlorophyta Chlamydomonas smithii Chlorophyta Chlamydomonas sp. Chlorophyta Chlamydomonas sphaeroides Chlorophyta Chlamydomonas subangulosa Chlorophyta Chlamydomonas surtseyiensis Chlorophyta Chlamydomonas toveli Chlorophyta Chlamydomonas ulvaensis Chlorophyta Chlamydomonas yellowstonensis Chlorophyta Chlamydomonas zebra Chlorophyta Chlamydomonas zimbabwiensis Chlorophyta Chloranomala cuprecola Chlorophyta Chlorella anitrata Chlorophyta Chlorella anitrata var. minor Chlorophyta Chlorella antarctica Chlorophyta Chlorella ap. Chlorophyta Chlorella autotrophica var. atypica Chlorophyta Chlorella capsulata Chlorophyta Chlorella fusca var. fusca Chlorophyta Chlorella fusca var. vacuolata Chlorophyta Chlorella glucotropha Chlorophyta Chlorella luteoviridis Chlorophyta Chlorella miniata Chlorophyta Chlorella nocturna Chlorophyta Chlorella parva Chlorophyta Chlorella regularis var. minima Chlorophyta Chlorella saccharophila Chlorophyta Chlorella saccharophila var. saccharophila Chlorophyta Chlorella sp. Chlorophyta Chlorella sphaerica Chlorophyta Chlorella stigmatophora Chlorophyta Chlorella vulgaris Chlorophyta Chlorella zofingiensis Chlorophyta Chlorochytrium lemnae Chlorophyta Chlorocladus australasicus Chlorophyta Chlorococcales Chlorophyta Chlorococcum acidum Chlorophyta Chlorococcum aegyptiacum Chlorophyta Chlorococcum aquaticum Chlorophyta Chlorococcum arenosum Chlorophyta Chlorococcum citriforme Chlorophyta Chlorococcum croceum Chlorophyta Chlorococcum diplobionticum Chlorophyta Chlorococcum echinozygotum Chlorophyta Chlorococcum elbense Chlorophyta Chlorococcum elkhartiense Chlorophyta Chlorococcum gelatinosum Chlorophyta Chlorococcum granulosum Chlorophyta Chlorococcum isabeliense Chlorophyta Chlorococcum lacustre Chlorophyta Chlorococcum loculatum Chlorophyta Chlorococcum microstigmatum Chlorophyta Chlorococcum nivale Chlorophyta Chlorococcum novaeangliae Chlorophyta Chlorococcum oleofaciens Chlorophyta Chlorococcum oviforme Chlorophyta Chlorococcum paludosum Chlorophyta Chlorococcum pamirum Chlorophyta Chlorococcum perforatum Chlorophyta Chlorococcum perplexum Chlorophyta Chlorococcum pinguideum Chlorophyta Chlorococcum pulchrum Chlorophyta Chlorococcum pyrenoidosum Chlorophyta Chlorococcum refringens Chlorophyta Chlorococcum reticulatum Chlorophyta Chlorococcum rugosum Chlorophyta Chlorococcum salsugineum Chlorophyta Chlorococcum sphacosum Chlorophyta Chlorococcum tatrense Chlorophyta Chlorococcum texanum Chlorophyta Chlorococcum typicum Chlorophyta Chlorococcum uliginosum Chlorophyta Chlorocystis kornmannii Chlorophyta Chlorocystis westii Chlorophyta Chlorogonium perforatum Chlorophyta Chlorogonium sp. Chlorophyta Chlorogonium tetragamum Chlorophyta Chlorogonium tetragamum Chlorophyta Chloromonas actinochloris Chlorophyta Chloromonas asteroidea Chlorophyta Chloromonas augustae Chlorophyta Chloromonas brevispina Chlorophyta Chloromonas carrizoensis Chlorophyta Chloromonas chenangoensis Chlorophyta Chloromonas clathrata Chlorophyta Chlorosarcinopsis Chlorophyta Chlorosarcinopsis amylophila Chlorophyta Chlorosarcinopsis arenicola Chlorophyta Chlorosarcinopsis auxotrophica Chlorophyta Chlorosarcinopsis bastropiensis Chlorophyta Chlorosarcinopsis deficiens Chlorophyta Chlorosarcinopsis dissociata Chlorophyta Chlorosarcinopsis eremi Chlorophyta Chlorosarcinopsis halophila Chlorophyta Chlorosarcinopsis minor Chlorophyta Chlorosarcinopsis negevensis f. ferruguinea

Chlorophyta Chlorosarcinopsis negevensis f. negevensis Chlorophyta Chlorosarcinopsis pseudominor Chlorophyta Chlorosarcinopsis sempervirens Chlorophyta Chlorosarcinopsis sp. Chlorophyta Chlorosarcinopsis variabilis Chlorophyta Coelastrum cambricum Chlorophyta Coelastrum proboscideum var. dilatatum Chlorophyta Coelastrum proboscideum var. gracile Chlorophyta Coelastrum sphaericum Chlorophyta Coenochloris planoconvexa Chlorophyta Cosmarium biretum Chlorophyta Cosmarium botrytis Chlorophyta Cosmarium connatum Chlorophyta Cosmarium cucumis Chlorophyta Cosmarium debaryi Chlorophyta Cosmarium formosulum Chlorophyta Cosmarium impressulum Chlorophyta Cosmarium margaritiferum Chlorophyta Cosmarium smolandicum Chlorophyta Cosmarium sp. Chlorophyta Cosmarium subcostatum Chlorophyta Cosmarium subtumidum Chlorophyta Cosmarium turpinii Chlorophyta Crucigenia lauterbornii Chlorophyta Crucigeniella rectangularis Chlorophyta Dictyococcus schumacherensis Chlorophyta Dictyococcus varians Chlorophyta Dictyosphaerium planctonicum Chlorophyta Diplostauron pentagonium Chlorophyta Gonium multicoccum Chlorophyta Gonium octonarium Chlorophyta Gonium quadratum Chlorophyta Gonium sacculiferum Chlorophyta Gonium sociale Chlorophyta Gonium sociale var. sacculum Chlorophyta Gonium sociale var. sociale Chlorophyta Gonium viridistellatum Chlorophyta Klebsormidium flaccidum var. cryophila Chlorophyta Klebsormidium marinum Chlorophyta Klebsormidium subtilissimum Chlorophyta Lagerheimia subsalsa Chlorophyta Mougeotia transeaui Chlorophyta Muriella aurantiaca Chlorophyta Muriella decolor Chlorophyta Mychonastes homosphaera Chlorophyta Nautococcus pyriformis Chlorophyta Nautococcus soluta Chlorophyta Neospongiococcum alabamense Chlorophyta Neospongiococcum butyrosum Chlorophyta Neospongiococcum commatiforme Chlorophyta Neospongiococcum concentricum Chlorophyta Neospongiococcum excentricum Chlorophyta Neospongiococcum giganticum Chlorophyta Neospongiococcum irregulare Chlorophyta Neospongiococcum macropyrenoidosum Chlorophyta Neospongiococcum mahleri Chlorophyta Neospongiococcum mobile Chlorophyta Neospongiococcum multinucleatum Chlorophyta Neospongiococcum proliferum Chlorophyta Neospongiococcum punctatum Chlorophyta Neospongiococcum rugosum Chlorophyta Neospongiococcum saccatum Chlorophyta Neospongiococcum solitarium Chlorophyta Neospongiococcum sphaericum Chlorophyta Neospongiococcum vacuolatum Chlorophyta Neospongiococcum variabile Chlorophyta Nephrochlamys subsolitaria Chlorophyta Oedogonium angustistomum Chlorophyta Oedogonium borisianum Chlorophyta Oedogonium calliandrum Chlorophyta Oedogonium cardiacum Chlorophyta Oedogonium donnellii Chlorophyta Oedogonium foveolatum Chlorophyta Oedogonium geniculatum Chlorophyta Oedogonium sp. Chlorophyta Oocystis alpina Chlorophyta Oocystis apiculata Chlorophyta Oocystis marssonii Chlorophyta Oocystis minuta Chlorophyta Oocystis sp Chlorophyta Pediastrum angulosum Chlorophyta Pediastrum boryanum var. cornutum Chlorophyta Pediastrum boryanum var. longicorne Chlorophyta Pediastrum clathratum Chlorophyta Pediastrum duplex var. asperum Chlorophyta Pediastrum simplex Chlorophyta Pediastrum sp. Chlorophyta Pithophora sp. Chlorophyta Pleurastrum erumpens Chlorophyta Pleurastrum terrestre Chlorophyta Pleurastrum terrestre var. indica Chlorophyta Protosiphon botryoides f. parieticola Chlorophyta Protosiphon sp. Chlorophyta Pseudendoclonium akinetum Chlorophyta Pseudendoclonium basiliensis Chlorophyta Pseudendoclonium prostratum Chlorophyta Pseudococcomyxa adhaerens Chlorophyta Raphidonema corcontica Chlorophyta Raphidonema longiseta Chlorophyta Raphidonema nivale Chlorophyta Raphidonema sp. Chlorophyta Raphidonema spiculiforme Chlorophyta Scenedesmus abundans Chlorophyta Scenedesmus arcuatus Chlorophyta Scenedesmus armatus Chlorophyta Scenedesmus basiliensis Chlorophyta Scenedesmus bijugatus var. seriatus Chlorophyta Scenedesmus breviaculeatus Chlorophyta Scenedesmus dispar Chlorophyta Scenedesmus hystrix Chlorophyta Scenedesmus jovais Chlorophyta Scenedesmus naegelii Chlorophyta Scenedesmus pannonicus Chlorophyta Scenedesmus parisiensis Chlorophyta Scenedesmus platydiscus Chlorophyta Scenedesmus sp. Chlorophyta Scenedesmus subspicatus Chlorophyta Selenastrum capricornutum Chlorophyta Selenastrum minutum Chlorophyta Selenastrum sp. Chlorophyta Sirogonium sticticum Chlorophyta Spirogyra condensata Chlorophyta Spirogyra crassispina Chlorophyta Spirogyra gracilis Chlorophyta Spirogyra grevilleana Chlorophyta Spirogyra juergensii Chlorophyta Spirogyra liana Chlorophyta Spirogyra maxima Chlorophyta Spirogyra meinningensis Chlorophyta Spirogyra notabilis Chlorophyta Spirogyra occidentalis Chlorophyta Spirogyra pratensis Chlorophyta Spirogyra quadrilaminata Chlorophyta Spirogyra rhizobrachialis

Chlorophyta Spirogyra sp.

Chlorophyta Spirogyra varians

Chlorophyta Stichococcus & Heterococcus spp.

Chlorophyta Stichococcus chodati

Chlorophyta Stichococcus fragilis

Chlorophyta Stichococcus mirabilis

Chlorophyta Stichococcus sequoieti

Chlorophyta Stigeoclonium aestivale

Chlorophyta Stigeoclonium farctum

Chlorophyta Stigeoclonium pascheri

Chlorophyta Stigeoclonium subsecundum

Chlorophyta Stigeoclonium tenue

Chlorophyta Stigeoclonium variabile

Chlorophyta Tetradesmus cumbricus

Chlorophyta Zygnema amosum

Chlorophyta Zygnema cylindricum

Chlorophyta Zygnema extenue

Chlorophyta Zygnema sp.

Chlorophyta Zygnema spontaneum

Chlorophyta Zygnema sterile

Cryptophyta Campylomonas reflexa

Cryptophyta Chroomonas coerulea

Cryptophyta Chroomonas diplococca

Cryptophyta Chroomonas pochmanii

Cryptophyta Chroomonas sp.

Cryptophyta Cryptochrysis sp.

Cryptophyta Cryptomonas ovata

Cryptophyta Cryptomonas ovata var. palustris

Cryptophyta Cryptomonas ozolini

Cryptophyta Cryptomonas sp.

Cryptophyta Hemiselmis sp.

Cryptophyta Proteomonas sulcata

Cryptophyta Rhodomonas salina

Cyanobacteria Anabaena aequalis

Cyanobacteria Anabaena catenula

Cyanobacteria Anabaena cylindrica

Cyanobacteria Anabaena flos-aquae

Cyanobacteria Anabaena inaequalis

Cyanobacteria Anabaena minutissima

Cyanobacteria Anabaena randhawae

Cyanobacteria Anabaena sp.

Cyanobacteria Anabaena sphaerica

Cyanobacteria Anabaena spiroides

Cyanobacteria Anabaena subcylindrica

Cyanobacteria Anabaena subtropica

Cyanobacteria Anabaena variabilis

Cyanobacteria Anabaena verrucosa Cyanobacteria Anacystis marina Cyanobacteria Aphanizomenon flos-aquae Cyanobacteria Arthrospira fusiformis Cyanobacteria Calothrix anomala Cyanobacteria Calothrix j avanica Cyanobacteria Calothrix membranacea Cyanobacteria Calothrix parietina Cyanobacteria Calothrix sp. Cyanobacteria Chamaesiphon sp. Cyanobacteria Chroococcidiopsis sp. Cyanobacteria Cylidrospermum sp. Cyanobacteria Cylindrospermopsis raciborskii Cyanobacteria Cylindrospermum licheniforme Cyanobacteria Cylindrospermum sp. Cyanobacteria Dermocarpa sp. Cyanobacteria Dermocarpa violacea Cyanobacteria Entophysalis sp. Cyanobacteria Eucapsis sp. Cyanobacteria Fischerella ambigua Cyanobacteria Fischerella muscicola Cyanobacteria Fremyella diplosiphon Cyanobacteria Gloeocapsa alpicola Cyanobacteria Gloeocapsa sp. Cyanobacteria Gloeotrichia echinulata Cyanobacteria Gloeotrichia ghosi Cyanobacteria Gloeotrichia sp. Cyanobacteria Hapalosiphon welwitschii Cyanobacteria Leptolyngbya nodulosa Cyanobacteria Lyngbya aestuarii Cyanobacteria Lyngbya kuetzingii Cyanobacteria Lyngbya lagerheimii Cyanobacteria Lyngbya purpurem Cyanobacteria Lyngbya sp. Cyanobacteria Mastigocladus laminosus Cyanobacteria Merismopedia glauca f. insignis Cyanobacteria Merismopedia sp. Cyanobacteria Microcoleus sp. Cyanobacteria Microcoleus vaginatus var. cyano-viridis Cyanobacteria Microcystis aeruginosa Cyanobacteria Microcystis flos-aquae Cyanobacteria Microcystis sp. Cyanobacteria Nodularia harveyana Cyanobacteria Nodularia spumigena Cyanobacteria Nostoc calcicola Cyanobacteria Nostoc commune Cyanobacteria Nostoc edaphicum Cyanobacteria Nostoc ellipsosporum Cyanobacteria Nostoc foliaceum Cyanobacteria Nostoc longstaffi Cyanobacteria Nostoc parmeloides Cyanobacteria Nostoc piscinale Cyanobacteria Nostoc punctiforme Cyanobacteria Nostoc sp. Cyanobacteria Nostoc zetterstedtii Cyanobacteria Oscillatoria amoena Cyanobacteria Oscillatoria animalis Cyanobacteria Oscillatoria borneti Cyanobacteria Oscillatoria brevis Cyanobacteria Oscillatoria lud Cyanobacteria Oscillatoria lutea Cyanobacteria Oscillatoria lutea var. contorta Cyanobacteria Oscillatoria prolifera Cyanobacteria Oscillatoria sp. Cyanobacteria Oscillatoria tenuis Cyanobacteria Phormidium autumnale Cyanobacteria Phormidium boneri Cyanobacteria Phormidium foveolarum Cyanobacteria Phormidium fragile Cyanobacteria Phormidium inundatum Cyanobacteria Phormidium luridum var. olivace Cyanobacteria Phormidium persicinum Cyanobacteria Phormidium sp. Cyanobacteria Plectonema boryanum Cyanobacteria Plectonema sp. Cyanobacteria Pleurocapsa uliginosa Cyanobacteria Porphyrosiphon notarisii Cyanobacteria Rubidibacter lacunae Cyanobacteria Schizothrix calcicola Cyanobacteria Schizothrix calcicola var. radiata Cyanobacteria Schizothrix calcicola var. vermiformis Cyanobacteria Scytonema Cyanobacteria Scytonema crispum Cyanobacteria Scytonema hofmanni Cyanobacteria Scytonema sp. Cyanobacteria Spirirestis rafaelensis Cyanobacteria Spirulina major Cyanobacteria Spirulina maxima Cyanobacteria Spirulina platensis Cyanobacteria Spirulina sp. Cyanobacteria Spirulina subsalsa Cyanobacteria Spirulina subsalsa f. versicolor Cyanobacteria Starria zimbabweensis Cyanobacteria Symphyonemopsis katniensis Cyanobacteria Symploca muscorum Cyanobacteria Synechococcus Cyanobacteria Synechococcus cedrorum Cyanobacteria Synechococcus elongatus

Cyanobacteria Synechococcus sp.

Cyanobacteria Synechocystis nigrescens

Cyanobacteria Synechocystis sp.

Cyanobacteria Tolypothrix distorta var. symplocoides

Dinophyta Amphidinium carterae

Dinophyta Amphidinium rhynchocephalum

Dinophyta Ceratocorys horrida

Dinophyta Gyrodinium dorsum

Dinophyta Heterocapsa niei

Dinophyta Heterocapsa pygmeae

Dinophyta Karenia brevis

Dinophyta Oxyrrhis marina

Dinophyta Peridinium foliaceum

Dinophyta Peridinium inconspicuum

Dinophyta Peridinium sociale

Dinophyta Prorocentrum cassubicum

Dinophyta Prorocentrum triestinum

Dinophyta Pyrocystis lunula

Dinophyta Pyrocystis noctiluca

Dinophyta Scrippsiella trochoidea

Dinophyta Zooxanthella microadriatica

Euglenozoa Colacium mucronatum

Euglenozoa Colacium vesiculosum

Euglenozoa Euglena acus var. gracilis

Euglenozoa Euglena anabaena

Euglenozoa Euglena cantabrica

Euglenozoa Euglena caudata

Euglenozoa Euglena deses

Euglenozoa Euglena geniculata var. terricola

Euglenozoa Euglena laciniata

Euglenozoa Euglena mutabilis

Euglenozoa Euglena myxocylindracea

Euglenozoa Euglena pisciformis var. obtusa

Euglenozoa Euglena proxima

Euglenozoa Euglena rubra

Euglenozoa Euglena sanguinea

Euglenozoa Euglena sp.

Euglenozoa Euglena spirogyra

Euglenozoa Euglena stellata

Euglenozoa Euglena terricola

Euglenozoa Euglena tripteris

Euglenozoa Eutreptia pertyi

Euglenozoa Lepocinclis buetschlii

Euglenozoa Lepocinclis ovata var. deflandriana

Euglenozoa Phacus acuminata

Euglenozoa Phacus brachykentron

Euglenozoa Phacus caudata Euglenozoa Phacus megalopsis

Euglenozoa Phacus pusillus

Euglenozoa Phacus triqueter

Euglenozoa Trachelomonas grandis

Euglenozoa Trachelomonas hispida

Euglenozoa Trachelomonas hispida var. coronata

Euglenozoa Trachelomonas oblonga var. punctata

Euglenozoa Trachelomonas volvocina

Euglenozoa Trachelomonas volvocinopsis var. spiralis

Glaucophyta Cyanophora biloba

Glaucophyta Cyanophora paradoxa

Glaucophyta Glaucocystis nostochinearum

Haptophyta Calyptrosphaera sphaeroidea

Haptophyta Chrysochromulina brevifilum

Haptophyta Coccolithophora sp.

Haptophyta Coccolithus neohelis

Haptophyta Cricosphaera carterae

Haptophyta Dicrateria inornata

Haptophyta Emiliania huxleyi

Haptophyta Isochrysis aff. galbana

Haptophyta Isochrysis galbana

Haptophyta Isochrysis sp.

Haptophyta Ochrosphaera neapolitana

Haptophyta Ochrosphaera verrucosa

Haptophyta Paylova gyrans

Haptophyta Pavlova lutheri

Haptophyta Pseudoisochrysis paradoxa

Haptophyta Sarcinochrysis marina

Oochrophyta Asterosiphon dichotomus

Oochrophyta Aureoumbra lagunensis

Oochrophyta Bodanella lauterborni

Oochrophyta Botrydiopsis arhiza

Oochrophyta Botrydium cystosum

Oochrophyta Bumilleria exilis

Oochrophyta Bumilleria sicula

Oochrophyta Bumilleriopsis sp.

Oochrophyta Chattonella japonica

Oochrophyta Chloridella miniata

Oochrophyta Chlorocloster solani

Oochrophyta Chlorocloster sp.

Oochrophyta Chromulina nebulosa

Oochrophyta Chrysochaete britannica

Oochrophyta Dictyopteris repens

Oochrophyta Dictyota cilliolata

Oochrophyta Dictyota dichotoma

Oochrophyta Dinobryon sp.

Oochrophyta Ectocaφus siliculosus

Oochrophyta Ectocarpus sp. Oochrophyta Ectocarpus variabilis Oochrophyta Ellipsoidion sp. Oochrophyta Epipyxis pulchra Oochrophyta Eustigmatos magna Oochrophyta Heterococcus caespitosus Oochrophyta Heterococcus cf. caespitosus Oochrophyta Heterococcus cf. endolithicus Oochrophyta Heterococcus cf. pleurococcoides Oochrophyta Heterococcus cf. protnematoides Oochrophyta Heterococcus chodati Oochrophyta Heterococcus fuornensis Oochrophyta Heterococcus mainxii Oochrophyta Heterococcus moniliformis Oochrophyta Heterococcus protonematoides Oochrophyta Heterococcus sp. Oochrophyta Heterococcus sp. Pleuroscoccoides Oochrophyta Heterothrix debilis Oochrophyta Heterotrichella gracilis Oochrophyta Hibberdia magna Oochrophyta Lagynion scherffelii Oochrophyta Mallomonas asmundae Oochrophyta Mischococcus sphaerocephalus Oochrophyta Monodus subterraneus Oochrophyta Nannochloropsis oculata Oochrophyta Ochromonas sp. Oochrophyta Ochromonas spherocystis Oochrophyta Ophiocytium maius Oochrophyta Phaeoplaca thallosa Oochrophyta Phaeoschizochlamys mucosa Oochrophyta Pleurochloris meiringensis Oochrophyta Pseudobumilleriopsis pyrenoidosa Oochrophyta Sorocarpus uvaeformis Oochrophyta Spermatochnus paradoxus Oochrophyta Sphacelaria cirrosa Oochrophyta Sphacelaria rigidula Oochrophyta Sphacelaria sp. Oochrophyta Stichogloea doederleinii Oochrophyta Synura petersenii Oochrophyta Synura uvella Oochrophyta Tribonema missouriense Oochrophyta Tribonema sp. Oochrophyta Vacuolaria virescens Oochrophyta Vaucheria bursata Oochrophyta Vaucheria geminata Oochrophyta Vaucheria sessilis Oochrophyta Vaucheria terrestris Oochrophyta Vischeria punctata Rhodophyta Acrochaetium flexuosum Rhodophyta Acrochaetium pectinatum Rhodophyta Acrochaetium plumosum Rhodophyta Acrochaetium proskaueri Rhodophyta Acrochaetium sagraeanum Rhodophyta Acrochaetium sp Rhodophyta Acrosorium uncinatum Rhodophyta Anfractutofilum umbracolens Rhodophyta Antithamnion defectum Rhodophyta Antithamnion glanduliferum Rhodophyta Apoglossum ruscifolium Rhodophyta Asterocytis ramosa Rhodophyta Asterocytis sp. Rhodophyta Audouinella eugenea Rhodophyta Audouinella hermannii Rhodophyta Bangia afusco-purpure Rhodophyta Bangia atro-purpurea Rhodophyta Bangia fusco-purpurea Rhodophyta Bangiopsis subsimplex Rhodophyta Batrachospermum intortum Rhodophyta Batrachospermum macrosporum Rhodophyta Batrachospermum moniliforme Rhodophyta Batrachospermum sirodotia Rhodophyta Batrachospermum sp. Rhodophyta Batrachospermum vagum var. keratophylum Rhodophyta Boldia erythrosiphon Rhodophyta Bostrychia bispora Rhodophyta Bostrychia tenella Rhodophyta Botryocladia ardreana Rhodophyta Botryocladia boergesenii Rhodophyta Botryocladia pyriformis Rhodophyta Bryothamnion triqutrum Rhodophyta Callithamnion baileyi Rhodophyta Callithamnion byssoides Rhodophyta Callithamnion corymbosum Rhodophyta Callithamnion halliae Rhodophyta Callithamnion paschale Rhodophyta Callithamnion roseum Rhodophyta Callithamnion sp. Rhodophyta Caloglossa intermedia Rhodophyta Caloglossa leprieurii f. pygmaea Rhodophyta Ceramium sp. Rhodophyta Champia parvula Rhodophyta Chondrus crispus Rhodophyta Compsopogon coeruleus Rhodophyta Compsopogon hooked Rhodophyta Compsopogon oishii Rhodophyta Compsopogonopsis leptoclados Rhodophyta Cumagloia andersonii Rhodophyta Cyanidium caldarium Rhodophyta Cystoclonium purpureum Rhodophyta Dasya pedicellata Rhodophyta Dasya rigidula Rhodophyta Digenea simplex Rhodophyta Dixoniella grisea Rhodophyta Erythrocladia sp. Rhodophyta Erythrotrichia carnea Rhodophyta Eupogodon planus Rhodophyta Flintiella sanguinaria Rhodophyta Gelidiopsis intricata Rhodophyta Glaucosphaera vacuolata Rhodophyta Gracilaria debilis Rhodophyta Gracilaria foliifera Rhodophyta Gracilaria verrucosa Rhodophyta Grateloupia filicina Rhodophyta Griffithsia pacifica Rhodophyta Heterosiphonia plumosa Rhodophyta Hildenbrandia prototypus Rhodophyta Hildenbrandia rivularis Rhodophyta Hypnea musciformis Rhodophyta Lomentaria articulata Rhodophyta Lomentaria orcadensis Rhodophyta Lophocladia trichoclados Rhodophyta Nemalion multifidum Rhodophyta Nemalionopsis shawi f. caroliniana Rhodophyta Nemalionopsis tortuosa Rhodophyta Neoagardhiella baileyi Rhodophyta Palmaria palmata Rhodophyta Phyllophora membranacea Rhodophyta Phyllophora truncata Rhodophyta Polyneura hilliae Rhodophyta Polyneura latissima Rhodophyta Polysiphonia boldii Rhodophyta Polysiphonia echinata Rhodophyta Porphyra eucosticta Rhodophyta Pseudochantransia sp. Rhodophyta Pterocladia americana Rhodophyta Pterocladia bartlettii Rhodophyta Pterocladia capillacea Rhodophyta Ptilothamnion sp. Rhodophyta Puφureofilum apyrenoidigerum Rhodophyta Rhodella maculata Rhodophyta Rhodochaete parvula Rhodophyta Rhodochorton puφureum Rhodophyta Rhodochorton tenue Rhodophyta Rhodosorus marinus Rhodophyta Rhodospora sordida

TABLE 4 - FURTHER EXAMPLES OF ALGAE STRAINS PRODUCING EXTRA- AND/OR INTRA-CELLULAR CELLULASE ENZYMES

ALGAE STRAINS

Division Genus/specie

Bacillariophyta Diadesmis gallica

Bacillariophyta Navicula atomus

Chlorophyta Actinastrum hantzschii

Chlorophyta Actinochloris sphaerica

Chlorophyta Ankistrodesmus spiralis

Chlorophyta Apatococcus lobatus

Chlorophyta Asterarcys cubensis

Chlorophyta Auxenochlorella protothecoides

Chlorophyta Botryococcus protuberans

Chlorophyta Botryococcus sudeticus

Chlorophyta Chaetophora cf. elegans

Chlorophyta Chantransia sp.

Chlorophyta Characium sieboldii

Chlorophyta Characium starrii

Chlorophyta Characium terrestre

Chlorophyta Chlamydomonas actinochloris

Chlorophyta Chlamydomonas agregata

Chlorophyta Chlamydomonas augustae

Chlorophyta Chlamydomonas cf. debaryana

Chlorophyta Chlamydomonas cf. peterfii

Chlorophyta Chlamydomonas cf. typica

Chlorophyta Chlamydomonas chlorococcoides

Chlorophyta Chlamydomonas dorsoventralis Chlorophyta Chlamydomonas geitleri Chlorophyta Chlamydomonas macropyrenoidosa Chlorophyta Chlamydomonas moewusii Chlorophyta Chlamydomonas nivalis Chlorophyta Chlamydomonas peterfii Chlorophyta Chlamydomonas segnis Chlorophyta Chlamydomonas subtilis Chlorophyta Chlorella cf. homosphaera Chlorophyta Chlorella homosphaera Chlorophyta Chlorella kessleri Chlorophyta Chlorella mirabilis Chlorophyta Chlorella sorokiniana Chlorophyta Chlorokybus atmophyticus Chlorophyta Chloromonas cf. paradoxa Chlorophyta Chloromonas jemtlandica Chlorophyta Chloromonas rosae Chlorophyta Chlorosarcinopsis aggregata Chlorophyta Chlorosarcinopsis gelatinosa Chlorophyta Chlorosarcinopsis minuta Chlorophyta Choricystis sp. Chlorophyta Coelastropsis costata Chlorophyta Coelastrum astroideum Chlorophyta Coelastrum microporum Chlorophyta Coelastrum morus Chlorophyta Coelastrum pseudomicroporum Chlorophyta Coelastrum reticulatum Chlorophyta Coenochloris pyrenoidosa Chlorophyta Coleochlamys cucumis Chlorophyta Cosmarium holmiense Chlorophyta Cosmarium meneghinii Chlorophyta Cosmarium subcrenatum Chlorophyta Crucigenia tetrapedia Chlorophyta Crucigeniella pulchra Chlorophyta Dictyococcus varians Chlorophyta Dictyosphaerium pulchellum Chlorophyta Dictyosphaerium tetrachotomum Chlorophyta Diplosphaera cf. chodatii Chlorophyta Enallax coelastroides Chlorophyta Enallax sp. Chlorophyta Geminella sp. Chlorophyta Gonium pectorale Chlorophyta Graesiella vacuolata Chlorophyta Interfilum paradoxum Chlorophyta Kentrosphaera austriaca Chlorophyta Kentrosphaera gibberosa Chlorophyta Keratococcus bicaudatus Chlorophyta Klebsormidium cf. scopulinum Chlorophyta Klebsormidium flaccidum Chlorophyta Klebsormidium pseudostichococcus Chlorophyta Klebsormidium rivulare Chlorophyta Klebsormidium sp. Chlorophyta Koliella sempervirens Chlorophyta Koliella spiculiformis Chlorophyta Lagerheimia marssonii Chlorophyta Lobosphaera sp. Chlorophyta Macrochloris radios a Chlorophyta Monoraphidium arcuatum Chlorophyta Monoraphidium cf. contortum Chlorophyta Monoraphidium contortum Chlorophyta Monoraphidium convolutum Chlorophyta Monoraphidium griffithii Chlorophyta Monoraphidium saxatile Chlorophyta Monoraphidium tortile Chlorophyta Mougeotia scalaris Chlorophyta Mougeotia sp. Chlorophyta Muriella sp. Chlorophyta Mychonastes sp. Chlorophyta Myrmecia bisecta Chlorophyta Nautococcus mammilatus Chlorophyta Nautococcus sp. Chlorophyta Neodesmus danubialis Chlorophyta Neospongiococcum granatum Chlorophyta Nephrochlamys rotunda Chlorophyta Oocystis cf. nephrocytioides Chlorophyta Oocystis lacustris Chlorophyta Pediastrum biradiatum Chlorophyta Pediastrum tetras Chlorophyta Pithophora roettleri Chlorophyta Pleurastrum paucicellulare Chlorophyta Pleurastrum sarcinoideum Chlorophyta Prasiolopsis ramosa Chlorophyta Protosiphon botryoides Chlorophyta Pseudendoclonium basiliense Chlorophyta Pseudendoclonium sp. Chlorophyta Pseudococcomyxa cf. simplex Chlorophyta Pseudococcomyxa simplex Chlorophyta Pseudococcomyxa sp. Chlorophyta Raphidocelis inclinata Chlorophyta Raphidocelis subcapitata Chlorophyta Raphidocelis valida Chlorophyta Raphidonema sempervirens Chlorophyta Rhexinema paucicellularis Chlorophyta Rhopalocystis cucumis Chlorophyta Scenedesmus cf. capitatus Chlorophyta Scenedesmus cf. ecornis Chlorophyta Scenedesmus cf. pseudoarmatus Chlorophyta Scenedesmus incrassatulus Chlorophyta Scenedesmus pecsensis Chlorophyta Scenedesmus pleiomorphus Chlorophyta Scenedesmus praetervisus Chlorophyta Schroederiella papillata

Chlorophyta Scotiella chlorelloidea

Chlorophyta Scotiellopsis oocystiformis

Chlorophyta Scotiellopsis reticulata

Chlorophyta Scotiellopsis albescens

Chlorophyta Scotiellopsis terrestris

Chlorophyta Selenastrum gracile

Chlorophyta Selenastrum rinoi

Chlorophyta Sphaerocystis bilobata

Chlorophyta Sphaerocystis schroeteri

Chlorophyta Spirogyra cf. semiornata

Chlorophyta Spirogyra communis

Chlorophyta Spirogyra lacustris

Chlorophyta Spirogyra mirabilis

Chlorophyta Spirogyra neglecta

Chlorophyta Stichococcus cf. chlorelloides

Chlorophyta Stichococcus chloranthus

Chlorophyta Stichococcus exiguus

Chlorophyta Stichococcus minutus

Chlorophyta Stichococcus sp.

Chlorophyta Stigeoclonium helveticum

Chlorophyta Stigeoclonium sp.

Chlorophyta Tetradesmus wisconsinensis

Chlorophyta Willea sp.

Chlorophyta Zygnema circumcarinatum

Chlorophyta Zygnema peliosporum

Cyanobacteria Bracteacoccus minor

Cyanobacteria Chlorococcum echinozygotum

Cyanobacteria Chlorococcum ellipsoideum

Cyanobacteria Chlorococcum hypnosporum

Cyanobacteria Chlorococcum infusiorum

Cyanobacteria Chlorococcum lobatum

Cyanobacteria Chlorococcum minutum

Cyanobacteria Chlorococcum scabellum

Cyanobacteria Chlorococcum vacuolatum

Cyanobacteria Chlorotetraedron bitridens

Cyanobacteria Chlorotetraedron incus

Cyanobacteria Chlorotetraedron polymorphum

Cyanobacteria Coccomyxa cf. gloeobotrydiformis

Cyanobacteria Coccomyxa glaronensis

Cyanobacteria Ettlia carotinosa

Cyanobacteria Fortiea rugulosa

Cyanobacteria Neochloris bilobata

Cyanobacteria Neochloris texensis

Cyanobacteria Neochloris vigensis

Cyanobacteria Spongiochloris spongiosa

Cyanobacteria Tetraedron caudatum

Cyanobacteria Tetraedron minimum

Cyanobacteria Tetrastrum komarekii

Euglenozoa Euglena gracilis var. urophora not assigned to a phylum Desmodesmus armatus not assigned to a phylum Desmodesmus brasiliensis not assigned to a phylum Desmodesmus cf. corallinus not assigned to a phylum Desmodesmus cf. gutwinskii not assigned to a phylum Desmodesmus cf. opoliensis var. mononensis not assigned to a phylum Desmodesmus cf. pannonicus not assigned to a phylum Desmodesmus cf. spinosus not assigned to a phylum Desmodesmus fuscus not assigned to a phylum Desmodesmus granulatus not assigned to a phylum Desmodesmus hirsutus not assigned to a phylum Desmodesmus quadricauda not assigned to a phylum Desmodesmus sempervirens not assigned to a phylum Desmodesmus subspicatus not assigned to a phylum Desmodesmus velitaris

Ochrophyta Botrydiopsis alpina

Ochrophyta Bumilleriopsis filiformis

Ochrophyta Bumilleriopsis peterseniana

Ochrophyta Chloridella neglecta

Ochrophyta Chloridella simplex

Ochrophyta Chlorobotrys regularis

Ochrophyta Ellipsoidion parvum

Ochrophyta Heterococcus brevicellularis

Ochrophyta Monodus guttula

Ochrophyta Monodus sp.

Ochrophyta Monodus subterraneus

Ochrophyta Nannochloropsis sp.

Ochrophyta Nephrodiella minor

Ochrophyta Pseudocharaciopsis ovalis

Ochrophyta Tribonema vulgare

Ochrophyta Vischeria helvetica

Ochrophyta Xanthonema bristolianum

Ochrophyta Xanthonema cf. debilis

Ochrophyta Xanthonema exile

Ochrophyta Xanthonema mucicolum

Ochrophyta Xanthonema sp.

Prasinophyta Dunaliella bioculata

Rhodophyta Microthamnion kuetzingianum

Rhodophyta Porphyridium aerugineum

Rhodophyta Porphyridium purpureum

Rhodophyta Porphyridium sordidum

Rhodophyta Porphyridium sp.