Patent Application
20110306101
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 21st century (Kerr R A, 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 CO2 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).
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:
(i) inoculating in a first bio-reactor a mixture of at least one of cellulose, hemicellulose, and lignin with at least one microorganism strain that produces one or more cellulases, hemicellulases, and/or laccases, that hydrolyze at least one of cellulose, hemicellulose and lignin, culturing said mixture under conditions to produce at least one fermentation product comprising one or more alcohols in said mixture;
(ii) optionally removing a portion of said at least one fermentation product comprising said one or more alcohols from said mixture;
(iii) transferring into a second bio-reactor all or the remaining portion of said mixture, wherein said remaining mixture contains all or a portion of said at least one fermentation product; and
(iv) inoculating the portion of said mixture containing said at least one fermentation product in said second bio-reactor with at least one algae strain that is capable of metabolizing said at least one fermentation product, and culturing said mixture under conditions so that said at least one algae strain produces one or more fatty acids.
The method can further comprise (v) recovering said one or more fatty acids from said at least one algae strain.
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 and alcohol production from biomass material. The fatty acids can be used, for example, in biofuel production.
One embodiment of the invention is directed to a method of producing fatty acids, by:
(i) inoculating in a first bio-reactor a mixture of at least one of cellulose, hemicellulose, and lignin with at least one microorganism strain that produces one or more cellulases, hemicellulases, and/or laccases that hydrolyze at least one of cellulose, hemicellulose and lignin and culturing the mixture to produce at least one fermentation product comprising one or more alcohols in said mixture;
(ii) optionally removing a portion of said one or more alcohols from said mixture,
(iii) transferring into a second bio-reactor the remaining portion of said remaining mixture, wherein said remaining mixture contains a portion of said at least one fermentation product; and
(iv) inoculating the portion of said mixture containing said at least one fermentation product in said second bio-reactor with at least one algae strain that metabolizes said at least one fermentation product, and culturing said mixture under conditions so that said at least one algae strain produces one or more fatty acids.
The method further includes (v) optionally recovering said one or more fatty acids from said at least one algae strain.
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 and pine are 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. Some other oil plant that carry a good yield in oil are poorly used as energy feedstock as their residual bean cake is toxic for mammal nutrition, like jatropha tree or castor bean plant, are actually good biomass crop. 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: about 5-35% lignin; about 10-35% hemicellulose; and about 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, husks, shells, grass, including switchgrass, miscanthus, hemp, vegetable pulp, corn, bean cake, corn stover, sugarcane, sugar beets, sorghum, cassaya, 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 can 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, H2SO4 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 et al., 1978; Hamilton et al., 1984) and showed enzyme hydrolysis could be greatly enhanced when the biomass structure is disrupted before hydrolysis Alkaline H2O2, ozone, organosolv (uses Lewis acids, FeCl3, (Al)2SO4 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 (H2SO4, HCl), ammonia-based solvents (NH3, 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 5¢/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, preferably an acid that 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 70° C. to 500° C., or in a range of about 120° C. to 200° C., or in a range of about 120° C. to 140° C.
Such acid pretreatment procedure can be used to generate the mixture utilized in step (i).
It should be noted that, when the biomass is obtained from plants, the mixture comprises at least one of cellulose, hemicellulose, lignin, furfural, phenolics and acetic acid.
After the pretreatment procedure, the mixture in the first bio-reactor comprises at least one of cellulose, hemicellulose, and lignin. In step (i), this mixture is inoculated with at least one microorganism strain that produces one or more cellulases, hemicellulases, and/or laccases that hydrolyze at least one of cellulose, hemicellulose and lignin to produce at least one fermentation product in said mixture.
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 (i). At the end of the enzymatic hydrolysis, the resultant medium can contain glucose, cellobiose, acetic acid, furfural, lignin, xylose, arabinose, rhamnose, mannose, galactose, and 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 (i). 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 in step (i).
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. According to still a further embodiment the extracellular and/or intracellular cellulase 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., and Clostridium lentocellum.
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 in step (i). 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., and Clostridium lentocellum.
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, and more preferably, an anaerobic bacterium, such as Clostridium lentocellum.
Again, any microorganism that is an extracellular and/or intracellular cellulase enzyme producer or extracellular and/or intracellular laccase enzyme producer can be utilized in the present invention to produce the requisite enzymes for enzymatic hydrolysis in step (i). Examples include those listed in attached 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 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, and 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 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 can have 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 of step (i). 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 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. Pat. No. 6,686,194-B1, 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 U.S. patent application Ser. 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 and acetic acid when using dilute acid 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 microorganisms 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 fungi strict or not.
In a further embodiment of the invention, the microorganism is a bacteria, and in particular, Clostridium lentocellum that has been evolutionarily modified by continuous culture.
In step (i), cellulase activity and/or the amount of fermentation products can be measured using common techniques, to determine the quantity of the fermentation product in the supernatant, before proceeding to step (iii).
It should be noted that the microorganism strain inoculated in the first bio-reactor catalyzes the cellulose into fermentation products (secondary metabolites) and soluble sugars such as xylose, arabinose, rhamnose, mannose, galactose, and other hemicellulose sugars that can then be used by the algae in step (iv) in the second bio-reactor.
The mixture from the first bio-reactor contains fermentation products and any sugar released not used by the microorganism strain from the reaction in the first bio-reactor. Such fermentation products can include acetate, acetone, 2,3-butanediol, butanol, butyrate, CO2, ethanol, formate, glycolate, lactate, malate, propionate, pyruvate, and succinate, and such released sugars can include glucose, cellobiose, xylose, mannose, arabinose, rhamnose, galactose and other hemicellulose sugars.
Step (ii) can optionally include an alcohol recovery step to remove/recover all or part of any alcohols, such as ethanol, butanol and others, produced in step (i).
Step (iii) of the invention involves transfer using common techniques, of the portion of the remaining mixture containing the at least one fermentation product into a second bio-reactor. Step (iii) can also include filtration, which involves separation of solid components and the first microorganism strain inoculated in the first bio-reactor from the fermentation products. Typically, this involves transfer of about 60-80% of soluble medium from the first bio-reactor to the second bio-reactor.
In one embodiment, at the start of step (ii) the mixture containing the fermentation products from step (i) is filtered with filters and transferred to another bio-reactor. For instance, an apparatus can be located for direct recovery of alcohol (for example by distillation) between the first and second bio-reactors. By utilizing conventional distillation/recovery techniques, one or more alcohols are partly or completely removed by a distillation process involving heating the bio-reactor. This allows for direct production/recovery of alcohol, such as ethanol and butanol.
The remaining mixture of fermentation products without the removed alcohol is then transferred to the second bio-reactor.
The soluble medium in the second bio-reactor thus contains fermentation products and any sugar released not used by the microorganism strain from the reaction in the first bio-reactor. Such fermentation products can include acetate, acetone, 2,3-butanediol, butanol, butyrate, CO2, ethanol, formate, glycolate, lactate, malate, propionate, pyruvate, and succinate, and such released sugars can include glucose, cellobiose, xylose, mannose, arabinose, rhamnose, galactose and other hemicellulose sugars.
Step (iv) of the invention involves inoculating the portion of said mixture containing said one or more fermentation products in the second bio-reactor with at least one algae strain that can metabolize the fermentation products and any of remaining soluble sugars such as xylose, arabinose, rhamnose, mannose, galactose, and other hemicelluloses sugars, and culturing the mixture under conditions so the algae produces one or more fatty acids.
In this step, one can utilize a phototrophic and/or heterotrophic algae and in aerobic and/or anaerobic environmental conditions. Such algae can use at least one of acetate, acetone, 2,3-butanediol, butanol, butyrate, CO2, ethanol, formate, glycolate, lactate, malate, propionate, pyruvate, and 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.
Preferably, the growth of said at least one algae strain is not substantially 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 of step (i), the algae in step (iii) 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, N.J., 1986, incorporated herein by reference), which can be grown in a closed bio-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 U.S. patent application Ser. No. 11/508,286, each incorporated herein by reference.
In doing so, 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 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 (iv) 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, and 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 step (iv) of 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 about 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, at least one algae strain in step (iv) produces no inhibitory by-product, for growth inhibition 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 at least one fermentation products, including acetic acid, ethanol, glucose, cellobiose, xylose or other hemicellulose sugars, pyruvate and succinate, under conditions so that algae strain produces one or more fatty acids.
By way of example, the algae utilized in step (iv) can be from the following taxonomic divisions of algae:
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);
B.
minor (Chodat) Petrova (1);
Pinguiococcus (8);
B.
terrestris (1);
Skeletonema (8);
Bracteacoccus sp. (1);
Emiliania (9);
Bumilleriopsis
brevis (5);
Prymnesium (9);
Chilomonas
paramecium (6);
Crypthecodinium (10);
Chlamydobotrys sp. (1);
Anabaenopsis
circularis (2);
Chlamydomonas
agloeformis (1);
Ankistrodesmus
braunii (1);
C.
dysosmos (1);
A.
falcatus (1);
C.
mundana Mojave strain
Botrydiopsis
intercedens (5);
Bracteacoccus
cinnabarinus (1);
C.
reinhardi (−) strain (1);
B.
engadiensis (1);
Chlorella
ellipsoidea (1);
C.
protothecoides (1);
N.
chlosterium (Ehr.) (3);
C.
pyrenoidosa (1);
N.
curvilineata Hust. (3);
C.
pyrenoidosa ATCC 7516 (1);
N.
filiformis (3);
C.
pyrenoidosa C-37-2 (1);
N.
frustulum (Kürtz.) (3);
C.
pyrenoidosa Emerson (1);
N.
laevis Hust. (3);
C.
pyrenoidosa 7-11-05 (1);
Nostoc
muscorum (2);
C.
vulgaris (1);
Ochromonas
malhamensis (4);
C.
vulgaris ATCC 9765 (1);
Pediastrum
boryanum (1);
C.
vulgaris Emerson (1);
P.
duplex (1);
C.
vulgaris Pratt-Trealease (1);
Polytoma
obtusum (1);
C.
vulgaris var. viridis (1);
P.
ocellatum (1);
Chlorellidium
tetrabotrys (5);
P.
uvella (1);
Chlorocloster
engadinensis (5);
Polytomella
caeca (or coeca) (1);
Chlorococcum
macrostigmatum (1);
Prototheca
zopfii (1);
Chlorococcum sp. (1);
Scenedesmus
acuminatus (1);
Chlorogloea
fritschii (2);
S.
acutiformis (1);
Chlorogonium
elongatum (1);
S.
costulatus Chod, var.
Coccomyxa
elongata (1);
chlorelloides (1);
Cyclotella sp. (3);
S.
dimorphus (1);
Dictyochloris
fragrans (1);
S.
obliquus (1);
Euglena
gracilis (7);
S.
quadricauda (1);
E.
gracilis Vischer (7);
Spongiochloris
excentrica (1);
E.
gracilis var. bacillaris (7);
S.
lamellate Deason (1);
E.
gracilis var. saccharophila (7);
S.
spongiosus (1);
Haematococcus
pluvialis (1);
Spongiochloris sp. (1);
Navicula
incerta Grun. (3);
Spongiococcum
alabamense (1);
N.
pelliculosa (3);
S.
excentricum (1);
Neochloris
alveolaris (1);
S.
excentricum Deason et Bold (1)
N.
aquatica Starr (1);
S.
multinucleatum (1);
N.
gelatinosa Herndon (1);
Stichococcus
bacillaris (1);
N.
pseudoalveolaris Deason (1);
S.
subtilis (1);
Neochloris sp. (1);
Tolypothrix
tenuis (2);
Nitzschia
angularis var.
Tribonema
aequale (5); and
affinis (3) (Grun.) perag.;
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 the attached 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 related to growing different types of algae and then harvesting algae for the purpose of producing biodiesel are incorporated herein by reference:
By employing the methods of the instant invention, the inoculation and culture of the mixture with the at least one algae strain in step (iv) results in the algae metabolizing at least one of glucose, cellobiose, xylose, mannose, galactose, rhamnose, arabinose or other hemicellulose sugars, and at least one of the fermentation products as acetate, acetone, 2,3-butanediol, butanol, butyrate, CO2, ethanol, formate, glycolate, lactate, malate, propionate, pyruvate, and succinate, under conditions so that said at least one algae strain produces one or more compounds, including fatty acids. In particular, the present invention in step (iv) 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 preferably contains 10 ml-50 ml per liter of refined glycerol, 0.1 ml-100 ml per liter of refined glycerol, and 2 ml-15 ml 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 preferably comprises 10-100% raw glycerol as a carbon source, 20-90% raw glycerol as a carbon source, 30-75% raw glycerol as a carbon source, 40-75% raw glycerol as a carbon source, or 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%, 15-85%, 20-80%, 25-75%, 35-50% 50-75%, and 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 one embodiment 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.
One exemplified embodiment of the method of the present invention can be found in the chart in
In 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° 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), (Fermentation) the mixture was inoculated with an evolutionarily modified microorganism strain of Clostridium lentocellum having the following properties and under the following conditions:
After the growth, enzymes production and fermentation product production phases, the mixture in the first bio-reactor in step (i) was subjected to filtration and the transfer step (iii), whereby the supernatant (containing mainly fermentation products produced in the first bio-reactor and some soluble sugars like xylose, arabinose, rhamnose, mannose, galactose, and other hemicelluloses sugars) was transferred to the second bio-reactor.
In step (iv) (i.e., the Conversion step), the supernatant from step (iii) (now in the second bio-reactor) was inoculated with an evolutionarily modified algae strain of Chlorella protothecoides (designated EVG16015) having the following properties and under the following conditions:
In step (iv), the algae were then grown under heterotrophic and aerobic conditions and 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 used supernatant from the second bio-reactor was reused by being reinjected into the first bio-reactor.
Another exemplified embodiment of the method of the present invention can be found in the chart in
In 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° 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), (Fermentation) the mixture was inoculated with an evolutionarily modified microorganism strain of Clostridium lentocellum having the following properties and under the following conditions:
After the growth, enzymes production and fermentation product production phases, the mixture in the first bio-reactor in step (i) was subjected to filtration and the transfer step (iii). In step (ii), a portion of the mixture containing the fermentation products from step (i) was filtered and transferred to another bio-reactor located between the first and second bio-reactors. Heat distillation was performed to remove alcohols, such as ethanol and butanol. The remaining mixture of fermentation products without the removed alcohol is then transferred to the second bio-reactor in step (iii).
This remaining mixture/soluble medium in the second bio-reactor contained fermentation products (no alcohol) and any sugar released not used by the microorganism strain from the reaction in the first bio-reactor. Such fermentation products (no alcohol like ethanol and butanol) included acetate, acetone, 2,3-butanediol, butanol, butyrate, CO2, ethanol, formate, glycolate, lactate, malate, propionate, pyruvate, and succinate, and such released sugars included glucose, cellobiose, xylose, mannose, arabinose, rhamnose, galactose and other hemicellulose sugars.
In step (iv) (i.e., the Conversion step), the remaining mixture (without the alcohol) from step (iii) (now in the second bio-reactor) was inoculated with an evolutionarily modified algae strain of Chlorella protothecoides (designated EVG16020) having the following properties and under the following conditions:
In step (iv), the algae were then grown under heterotrophic and aerobic conditions and 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 used supernatant from the second bio-reactor was reused by being reinjected into the first bio-reactor.
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.
Caldivirga maquilingensis
Sulfolobus acidocaldarius
Sulfolobus solfataricus
Thermofilum pendens
Picrophilus torridus
Pyrococcus abyssi
Pyrococcus furiosus
Pyrococcus horikoshii
Thermoplasma volcanium
Acidobacterium capsulatum
Acidothermus cellulolyticus
Actinomadura sp.
Actinomyces sp.
Amycolatopsis orientalis
Arthrobacter aurescens
Arthrobacter sp.
Bifidobacterium adolescentis
Bifidobacterium animalis
Bifidobacterium bifidum
Bifidobacterium longum
Cellulomonas fimi
Cellulomonas flavigena
Cellulomonas pachnodae
Cellulomonas uda
Cellulosimicrobium sp.
Clavibacter michiganensis subsp. michiganensis
Clavibacter michiganensis subsp. sepedonicus
Frankia alni
Frankia sp.
Jonesia sp.
Kineococcus radiotolerans
Leifsonia xyli subsp. xyli
Microbispora bispora
Micromonospora cellulolyticum
Mycobacterium abscessus
Mycobacterium avium
Mycobacterium avium subsp. Paratuberculosis
Mycobacterium bovis
Mycobacterium gilvum
Mycobacterium marinum
Mycobacterium smegmatis
Mycobacterium sp.
Mycobacterium tuberculosis
Mycobacterium ulcerans
Mycobacterium vanbaalenii
Mycobacterium vanbaalenii
Nocardioides sp.
Propionibacterium acnes
Rhodococcus equi
Saccharopolyspora erythraea
Saccharothrix australiensis
Salinispora arenicola
Salinispora tropica
Streptomyces ambofaciens
Streptomyces avermitilis
Streptomyces chartreusis
Streptomyces chattanoogensis
Streptomyces coelicolor
Streptomyces fradiae var.
Streptomyces griseus
Streptomyces griseus subsp. griseus
Streptomyces halstedii
Streptomyces lividans
Streptomyces nanchangensis
Streptomyces olivaceoviridis
Streptomyces reticuli
Streptomyces roseiscleroticus
Streptomyces sp.
Streptomyces thermocyaneoviolaceus
Streptomyces thermoviolaceus
Streptomyces turgidiscabies
Streptomyces viridosporus
Thermobifida alba
Thermobifida fusca
Thermopolyspora flexuosa
Bacteroides cellulosolvens
Bacteroides fragilis
Bacteroides ovatus
Bacteroides thetaiotaomicron
Bacteroides vulgatus
Cytophaga hutchinsonii
Cytophaga xylanolytica
Flavobacterium johnsoniae
Flavobacterium psychrophilum
Flavobacterium sp.
Gramella forsetii
Parabacteroides distasonis
Prevotella bryantii
Prevotella ruminicola
Rhodothermus marinus
Chlorobium chlorochromatii
Pelodictyon luteolum
Chloroflexus aurantiacus
Herpetosiphon aurantiacus
Roseiflexus castenholzii
Roseiflexus sp.
Anabaena variabilis
Nostoc punctiforme
Nostoc sp.
Synechococcus elongatus
Synechococcus sp.
Synechocystis sp.
Deinococcus geothermalis
Thermus caldophilus
Dictyoglomus thermophilum
Fibrobacter intestinalis
Fibrobacter succinogenes
Fibrobacter succinogenes subsp. succinogenes
Acetivibrio cellulolyticus
Alicyclobacillus acidocaldarius
Alkaliphilus metalliredigens
Anoxybacillus kestanbolensis
Bacillus agaradhaerens
Bacillus alcalophilus
Bacillus amyloliquefaciens
Bacillus anthracis
Bacillus cereus
Bacillus circulans
Bacillus clausii
Bacillus firmus
Bacillus halodurans
Bacillus licheniformis
Bacillus plakortiensis
Bacillus pumilus
Bacillus sp.
Bacillus subtilis
Bacillus subtilis subsp. subtilis
Bacillus thuringiensis serovar alesti
Bacillus thuringiensis serovar canadensis
Bacillus thuringiensis serovar darmstadiensis
Bacillus thuringiensis serovar israelensis
Bacillus thuringiensis serovar morrisoni
Bacillus thuringiensis serovar san diego
Bacillus thuringiensis serovar sotto
Bacillus thuringiensis serovar thompsoni
Bacillus thuringiensis serovar tochigiensis
Butyrivibrio fibrisolvens
Caldicellulosiruptor saccharolyticus
Caldicellulosiruptor sp.
Clostridium acetobutylicum
Clostridium beijerinckii
Clostridium cellulolyticum
Clostridium cellulovorans
Clostridium difficile
Clostridium josui
Clostridium lentocellum
Clostridium longisporum
Clostridium phytofermentans
Clostridium phytofermentans
Clostridium saccharobutylicum
Clostridium sp.
Clostridium stercorarium
Clostridium thermocellum
Eubacterium cellulosolvens
Eubacterium ruminantium
Geobacillus caldoxylosilyticus
Geobacillus stearothermophilus
Geobacillus thermodenitrificans
Geobacillus thermoleovorans
Lactobacillus acidophilus
Lactobacillus brevis
Lactobacillus gasseri
Lactobacillus johnsonii
Lactobacillus reuteri
Lactococcus lactis subsp. cremoris
Lactococcus lactis subsp. lactis
Leuconostoc mesenteroides subsp. Mesenteroides
Listeria innocua
Listeria monocytogenes
Paenibacillus barcinonensis
Paenibacillus curdlanolyticus
Paenibacillus fukuinensis
Paenibacillus lautus
Paenibacillus pabuli
Paenibacillus polymyxa
Paenibacillus sp.
Ruminococcus albus
Ruminococcus flavefaciens
Streptococcus mutans
Streptococcus sanguinis
Syntrophomonas wolfei subsp. wolfei
Thermoanaerobacter pseudethanolicus
Thermoanaerobacter sp.
Thermoanaerobacter tengcongensis
Thermoanaerobacterium polysaccharolyticum
Thermoanaerobacterium saccharolyticum
Thermoanaerobacterium sp.
Thermoanaerobacterium thermosulfurigenes
Thermobacillus xylanilyticus
Fusobacterium mortiferum
Rhodopirellula baltica
Acidiphilium cryptum
Acidovorax avenae subsp. citrulli
Acinetobacter baumannii
Aeromonas hydrophila
Aeromonas hydrophila subsp. hydrophila
Aeromonas punctata
Aeromonas salmonicida subsp. salmonicida
Agrobacterium tumefaciens
Alcaligenes sp.
Anaeromyxobacter dehalogenans
Anaeromyxobacter sp.
Asaia bogorensis
Azoarcus sp.
Azorhizobium caulinodans
Beijerinckia indica subsp. indica
Bordetella avium
Bradyrhizobium japonicum
Brucella abortus
Brucella canis
Brucella melitensis
Brucella ovis
Brucella suis
Burkholderia ambifaria
Burkholderia ambifaria
Burkholderia cenocepacia
Burkholderia cepacia
Burkholderia mallei
Burkholderia multivorans
Burkholderia phymatum
Burkholderia phytofirmans
Burkholderia pseudomallei
Burkholderia sp.
Burkholderia sp.
Burkholderia thailandensis
Burkholderia vietnamiensis
Burkholderia xenovorans
Caulobacter crescentus
Caulobacter sp.
Cellvibrio japonicus (formerly Pseudomonas cellulosa)
Cellvibrio mixtus
Chromobacterium violaceum
Citrobacter koseri
Colwellia psychrerythraea
Enterobacter cloacae
Enterobacter cloacae
Enterobacter sakazakii
Enterobacter sp.
Erwinia carotovora
Erwinia carotovora subsp. Atroseptica
Erwinia chrysanthemi
Erwinia rhapontici
Erwinia tasmaniensis
Escherichia coli
Gluconacetobacter diazotrophicus
Gluconacetobacter xylinus
Hahella chejuensis
Halorhodospira halophila
Klebsiella pneumoniae
Klebsiella pneumoniae subsp. pneumoniae
Legionella pneumophila Lens
Legionella pneumophila Paris
Legionella pneumophila str. Corby
Legionella pneumophila subsp. Pneumophila
Leptothrix cholodnii
Leptothrix cholodnii
Lysobacter sp.
Maricaulis maris
Marinomonas sp.
Mesorhizobium loti
Methylobacillus flagellatus
Methylobacterium extorquens
Methylobacterium radiotolerans
Methylobacterium sp.
Myxococcus xanthus
Nitrosospira multiformis
Parvibaculum lavamentivorans
Pectobacterium carotovorum
Pectobacterium carotovorum atroseptica
Pectobacterium carotovorum subsp. carotovorum
Photobacterium profundum
Polaromonas sp.
Polynucleobacter sp.
Proteus mirabilis
Pseudoalteromonas atlantica
Pseudoalteromonas atlantica
Pseudoalteromonas haloplanktis
Pseudoalteromonas sp.
Pseudomonas entomophila
Pseudomonas fluorescens
Pseudomonas putida
Pseudomonas sp.
Pseudomonas stutzeri
Pseudomonas syringae pv. mori
Pseudomonas syringae pv. phaseolicola
Pseudomonas syringae pv. syringae
Pseudomonas syringae pv. Tomato
Psychromonas ingrahamii
Ralstonia eutropha
Ralstonia metallidurans
Ralstonia solanacearum
Ralstonia syzygii
Rhizobium etli
Rhizobium leguminosarum bv. trifolii
Rhizobium sp.
Rhodobacter sphaeroides
Rhodoferax ferrireducens
Rhodopseudomonas palustris
Saccharophagus degradans
Salmonella enterica subsp. arizonae
Salmonella typhimurium
Serratia proteamaculans
Shigella boydii
Shigella flexneri
Shigella sonnei
Sinorhizobium medicae
Sinorhizobium meliloti
Sorangium cellulosum
Stigmatella aurantiaca
Teredinibacter turnerae
Thiobacillus denitrificans
Vibrio cholerae
Vibrio fischeri
Vibrio harveyi
Vibrio parahaemolyticus
Vibrio sp.
Vibrio vulnificus
Xanthomonas albilineans
Xanthomonas axonopodis pv. citri str.
Xanthomonas campestris pv. campestris
Xanthomonas campestris pv. vesicatoria
Xanthomonas oryzae pv. oryzae
Xylella fastidiosa
Yersinia enterocolitica subsp. enterocolitica
Yersinia enterocolitica subsp. enterocolitica
Yersinia pestis
Yersinia pestis
Yersinia pestis Antiqua
Yersinia pestis biovar Medievalis
Yersinia pseudotuberculosis
Yersinia pseudotuberculosis
Zymomonas mobilis subsp. mobilis
Leptospira biflexa
Leptospira borgpetersenii
Leptospira interrogans
Fervidobacterium nodosum
Petrotoga mobilis
Thermotoga lettingae
Thermotoga maritima
Thermotoga neapolitana
Thermotoga petrophila
Thermotoga sp.
Opitutus terrae
Acremonium cellulolyticus
Acremonium sp.
Acremonium thermophilum
Alternaria alternata
Aspergillus aculeatus
Aspergillus flavus
Aspergillus fumigatus
Aspergillus kawachii
Aspergillus nidulans
Aspergillus niger
Aspergillus oryzae
Aspergillus sojae
Aspergillus sp.
Aspergillus sulphureus
Aspergillus terreus
Aspergillus tubingensis
Aspergillus versicolor
Aureobasidium pullulans var. melanigenum
Beltraniella portoricensis
Bionectria ochroleuca
Blumeria graminis
Botryosphaeria rhodina
Botryotinia fuckeliana
Candida albicans
Candida glabrata
Candida oleophila
Chaetomidium pingtungium
Chaetomium brasiliense
Chaetomium thermophilum
Chaetomium thermophilum var. thermophilum
Chrysosporium lucknowense
Claviceps purpurea
Coccidioides posadasii
Cochliobolus heterostrophus
Coniothyrium minitans
Corynascus heterothallicus
Cryphonectria parasitica
Cryptovalsa sp.
Cylindrocarpon sp.
Daldinia eschscholzii
Debaryomyces hansenii
Debaryomyces occidentalis
Emericella desertorum
Emericella nidulans
Epichloe festucae
Eremothecium gossypii
Fusarium anguioides
Fusarium chlamydosporum
Fusarium culmorum
Fusarium equiseti
Fusarium lateritium
Fusarium oxysporum
Fusarium poae
Fusarium proliferatum
Fusarium sp.
Fusarium tricinctum
Fusarium udum
Fusarium venenatum
Fusicoccum sp.
Geotrichum sp.
Gibberella avenacea
Gibberella moniliformis
Gibberella pulicaris
Gibberella zeae
Gliocladium catenulatum
Humicola grisea
Humicola grisea var. thermoidea
Humicola insolens
Humicola nigrescens
Hypocrea jecorina
Hypocrea koningii
Hypocrea lixii
Hypocrea pseudokoningii
Hypocrea schweinitzii
Hypocrea virens
Kluyveromyces lactis
Lacazia loboi
Leptosphaeria maculans
Macrophomina phaseolina
Magnaporthe grisea
Malbranchea cinnamomea
Melanocarpus
Melanocarpus albomyces
Nectria haematococca
Nectria ipomoeae
Neotyphodium lolii
Neotyphodium sp.
Neurospora crassa
Nigrospora sp.
Paecilomyces lilacinus
Paracoccidioides brasiliensis (various strains)
Penicillium canescens
Penicillium chrysogenum
Penicillium citrinum
Penicillium decumbens
Penicillium funiculosum
Penicillium janthinellum
Penicillium occitanis
Penicillium oxalicum
Penicillium purpurogenum
Penicillium simplicissimum
Pichia angusta
Pichia anomala
Pichia guilliermondii
Pichia pastoris
Pichia stipitis
Pseudoplectania nigrella
Robillarda sp.
Saccharomyces bayanus
Saccharomyces castellii
Saccharomyces cerevisiae
Saccharomyces kluyveri
Saccobolus dilutellus
Sarcoscypha occidentalis
Schizosaccharomyces pombe
Scopulariopsis brevicaulis
Scytalidium thermophilum
Stachybotrys chartarum
Stachybotrys echinata
Staphylotrichum coccosporum
Stilbella annulata
Talaromyces emersonii
Thermoascus aurantiacus
Thermoascus aurantiacus var. levisporus
Thermomyces lanuginosus
Thermomyces verrucosus
Thielavia australiensis
Thielavia microspora
Thielavia terrestris
Trichoderma asperellum
Trichoderma longibrachiatum
Trichoderma parceramosum
Trichoderma sp.
Trichoderma viride
Trichophaea saccata
Trichothecium roseum
Verticillium dahliae
Verticillium fungicola
Verticillium tenerum
Volutella colletotrichoides
Xylaria polymorpha
Yarrowia lipolytica
Agaricus bisporus
Armillariella tabescens
Athelia rolfsii
Chlorophyllum molybdites
Clitocybe nuda
Clitopilus prunulus
Coprinopsis cinerea
Crinipellis stipitaria
Cryptococcus adeliensis
Cryptococcus flavus
Cryptococcus neoformans
Cryptococcus neoformans var. neoformans
Cryptococcus sp.
Exidia glandulosa
Filobasidium floriforme (Cryptococcus albidus)
Fomitopsis palustris
Gloeophyllum sepiarium
Gloeophyllum trabeum
Infundibulicybe gibba
Irpex lacteus
Lentinula edodes
Meripilus giganteus
Phanerochaete chrysosporium
Pleurotus sajor-caju
Pleurotus sp.
Polyporus arcularius
Schizophyllum commune
Trametes hirsuta
Trametes versicolor
Ustilago maydis
Volvariella volvacea
Xylaria hypoxylon
Chlorella vulgaris
Anaeromyces sp.
Neocallimastix frontalis
Neocallimastix patriciarum
Neocallimastix sp.
Orpinomyces joyonii
Orpinomyces sp.
Hydra magnipapillata
Dictyostelium discoideum
Eisenia andrei
Phytophthora cinnamomi
Phytophthora infestans
Phytophthora ramorum
Phytophthora sojae
Ostreococcus lucimarinus
Ostreococcus tauri
Mucor circinelloides
Phycomyces nitens
Poitrasia circinans
Rhizopus oryzae
Syncephalastrum racemosum
Alternaria alternata
Arxula adeninivorans
Ashbya gossypii
Aspergillus fumigatus
Aspergillus niger
Aspergillus oryzae
Aspergillus terreus
Botryotinia fuckeliana
Buergenerula spartinae
Candida albicans
Candida glabrata
Chaetomium globosum
Chaetomium thermophilum var.
thermophilum
Claviceps purpurea
Coccidioides immitis
Colletotrichum lagenarium
Corynascus heterothallicus
Cryphonectria parasitica
Cryptococcus bacillisporus
Cryptococcus gattii
Cryptococcus neoformans
Cryptococcus neoformans var. neoformans
Davidiella tassiana
Debaryomyces hansenii
Emericella nidulans
Fusarium oxysporum
Fusarium oxysporum f. sp. lycopersici
Fusarium proliferatum
Gaeumannomyces graminis
Gaeumannomyces graminis var. graminis
Gaeumannomyces graminis var. tritici
Gibberella zeae
Glomerella cingulata
Hortaea acidophila
Humicola insolens
Hypomyces rosellus
Hypoxylon sp.
Kluyveromyces lactis
Lachnum spartinae
Lactarius blennius
Lactarius subdulcis
Melanocarpus albomyces
Morchella conica
Morchella crassipes
Morchella elata
Morchella esculenta
Morchella sp.
Morchella spongiola
Mycosphaerella sp.
Neurospora crassa
Paracoccidioides brasiliensis
Penicillium adametzii
Penicillium amagasakiense
Penicillium expansum
Penicillium simplissimum
Penicillium variabile
Phaeosphaeria halima
Phaeosphaeria spartinicola
Pichia pastoris
Pleospora spartinae
Podospora anserina
Saccharomyces cerevisiae
Saccharomyces pastorianus
Schizosaccharomyces pombe
Stagonospora sp.
Talaromyces flavus
Verpa conica
Yarrowia lipolytica
Agaricus bisporus
Amanita citrina
Amylostereum areolatum
Amylostereum chailletii
Amylostereum ferreum
Amylostereum laevigatum
Amylostereum sp.
Athelia rolfsii
Auricularia auricula-judae
Auricularia polytricha
Bjerkandera adusta
Bjerkandera sp.
Bondarzewia montana
Ceriporiopsis rivulosa
Ceriporiopsis subvermispora
Cerrena unicolor
Climacocystis borealis
Clitocybe nebularis
Clitocybe quercina
Collybia butyracea
Coniophora puteana
Coprinellus congregatus
Coprinellus disseminatus
Coprinopsis cinerea
Coprinopsis cinerea okayama
Coriolopsis gallica
Cortinarius flexipes
Crinipellis sp.
Cyathus bulleri
Cyathus sp.
Daedalea quercina
Dichomitus squalens
Echinodontium japonicum
Echinodontium tinctorium
Echinodontium tsugicola
Filobasidiella neoformans
Flammulina velutipes
Funalia trogii
Ganoderma applanatum
Ganoderma australe
Ganoderma formosanum
Ganoderma lucidum
Ganoderma sp.
Ganoderma tsunodae
Gloeophyllum trabeum
Grifola frondosa
Gymnopus fusipes
Gymnopus peronatus
Gyromitra esculenta
Halocyphina villosa
Hebeloma radicosum
Heterobasidion abietinum
Heterobasidion annosum
Heterobasidion araucariae
Heterobasidion insulare
Heterobasidion parviporum
Hypholoma sp.
Irpex lacteus
Lentinula edodes
Lentinus tigrinus
Lepista flaccida
Lepista irina
Lepista nuda
Lyophyllum shimeji
Macrolepiota procera
Macrotyphula juncea
Malassezia sympodialis
Marasmius alliaceus
Megacollybia platyphylla
Mycena cinerella
Mycena crocata
Mycena galopus
Mycena rosea
Mycena zephirus
Panus rudis
Panus sp.
Paxillus involutus
Peniophora sp.
Phanerochaete chrysosporium
Phanerochaete flavidoalba
Phanerochaete sordida
Phlebia radiata
Phlebiopsis gigantea
Piloderma byssinum
Piriformospora indica
Pleurotus cornucopiae
Pleurotus eryngii
Pleurotus ostreatus
Pleurotus pulmonarius
Pleurotus sajor-caju
Pleurotus sapidus
Pleurotus sp. ‘Florida’
Polyporus alveolaris
Polyporus ciliatus
Psathyrella corrugis
Psathyrella dicrani
Psathyrella murcida
Pycnoporus cinnabarinus
Pycnoporus coccineus
Pycnoporus sanguineus
Rigidoporus microporus
Russula atropurpurea
Russula mairei
Russula nigricans
Russula ochroleuca
Schizopora paradoxa
Schizophyllum commune
Schizophyllum commune f. trop. radiatum
Spongipellis sp.
Stropharia squamosa
Termitomyces sp.
Thanatephorus cucumeris
Trametes cervina
Trametes hirsuta
Trametes ochracea
Trametes pubescens
Trametes sp.
Trametes versicolor
Trametes villosa
Ustilago maydis
Volvariella volvacea
Xerocomus chrysenteron
Xylaria sp.
Achnanthes coarctata
Achnanthes inflata
Achnanthidium biporomum
Achnanthidium exiguum
Achnanthidium lanceolatum
Achnanthidium minutissimum
Achnanthidium rostratum
Amphora coffeaeformis
Amphora coffeiformis
Amphora commutata
Amphora montana
Amphora pediculus
Amphora veneta
Anomoeoneis fogedii
Anomoeoneis sphaerophora
Anomoeoneis sphaerophora f. costata
Asterionella formosa
Aulacoseira ambigua
Aulacoseira granulata
Bacillaria paxillifer
Caloneis bacillum
Caloneis lewisii
Caloneis molaris
Caloneis ventricosa
Campylodiscus clypeus
Chaetoceros elmorei
Chaetoceros gracilis
Chaetoceros muelleri
Cocconeis placentula var. lineata
Craticula accomoda
Craticula cuspidata
Craticula halophila
Ctenophora pulchella
Cyclotella choctawatcheeana
Cyclotella meneghiniana
Cyclotella quillensis
Cylindrotheca fusiformis
Cylindrotheca gracilis
Cymatopleura elliptica
Cymatopleura librile
Cymbella aspera
Cymbella cistula
Cymbella microcephala
Cymbella norvegica
Cymbella pusilla
Cymbella tumida
Denticula kuetzingii
Diadesmis confervacea
Diatoma tenue var. elongatum
Diploneis subovalis
Encyonema minutum var. pseudogracilis
Entomoneis paludosa
Eucocconeis sp.
Eunotia curvata
Eunotia flexulosa
Eunotia formica
Eunotia glacialis
Eunotia maior
Eunotia naegelii
Eunotia pectinalis
Eunotia sp.
Fallacia monoculata
Fallacia pygmaea
Fragilaria capucina
Fragilaria crotonensis
Fragilariforma virescens
Gomphonema affine
Gomphonema affine var. insigne
Gomphonema angustatum
Gomphonema brebissonii
Gomphonema carolinense
Gomphonema dichotomum
Gomphonema gracile
Gomphonema intracatum
Gomphonema intracatum var. vibrio
Gomphonema parvulum
Gomphonema subclavatum var. commutatum
Gomphonema subclavatum var. mexicanum
Gomphonema subtile
Gomphonema truncatum
Gyrosigma acuminatum
Gyrosigma obtusatum
Gyrosigma spencerii var. curvula
Hantzschia amphioxys
Hantzschia amphioxys f. capitata
Hantzschia amphioxys var. maior
Hantzschia elongata
Hantzschia sigma
Hantzschia spectabilis
Hantzschia virgata var. gracilis
Lemnicola hungarica
Minutocellis sp.
Navicula abiskoensis
Navicula angusta
Navicula arvensis
Navicula capitata
Navicula cincta
Navicula cryptocephala
Navicula cryptocephala var. veneta
Navicula decussis
Navicula erifuga
Navicula gerloffii
Navicula incerta
Navicula libonensis
Navicula menisculus var. upsaliensis
Navicula minima
Navicula minima var. atomoides
Navicula phyllepta
Navicula radiosa
Navicula radiosa f. tenella
Navicula radiosa var. tenella
Navicula recens
Navicula reinhardtii
Navicula rhynchocephala var. amphiceros
Navicula salinarum
Navicula secura
Navicula seminuloides
Navicula seminulum
Navicula subrhynchocephala
Navicula tantula
Navicula tenelloides
Navicula tripunctata
Navicula tripunctata var. schizonemoides
Navicula trivialis
Navicula viridula var. rostellata
Neidium affine
Neidium affine var. humerus
Neidium affine var. longiceps
Neidium affine var. undulatum
Neidium affine var. undulatum
Neidium bisulcatum
Neidium bisulcatum var. subampilatum
Neidium productum
Nitzschia acicularis
Nitzschia amphibia
Nitzschia amphibioides
Nitzschia communis
Nitzschia commutata
Nitzschia dissipata
Nitzschia gracilis
Nitzschia linearis
Nitzschia linearis var. tenuis
Nitzschia nana
Nitzschia ovalis
Nitzschia paleacea
Nitzschia perminuta
Nitzschia reversa
Nitzschia rostellata
Nitzschia sigma
Nitzschia sp.
Nitzschia subtilioides
Nitzschia terricola
Nitzschia vermicularis
Nitzschia vitrea
Orthoseira dendroteres
Phaeodactylum tricornutum
Pinnularia appendiculata
Pinnularia biceps
Pinnularia borealis
Pinnularia brebissonii
Pinnularia gibba
Pinnularia mayeri
Pinnularia mesolepta
Pinnularia nodosa
Pinnularia sp.
Pinnularia subcapitata
Pinnularia subcapitata var. Elongata
Pinnularia subgibba
Pinnularia termitina
Pinnularia viridiformis
Placoneis clementis
Placoneis elginensis
Pleurosigma elongatum
Pleurosira laevis
Pseudostaurosira construens
Rhopalodia contorta
Rhopalodia gibba
Scoliopleura peisonis
Sellaphora pupula
Sellaphora pupula var. rectangularis
Skeletonema costatum
Stauroneis acuta
Stauroneis anceps
Stauroneis anceps f. gracilis
Stauroneis anceps var. gracilis
Stauroneis phoenicenteron
Stauroneis phoenicenteron f. gracilis
Stauroneis smithii var. incisa
Staurosira construens
Staurosirella pinnata
Stenopterobia curvula
Stephanodiscus minutulus
Stephanodiscus parvus
Surirella angusta
Surirella brightwellii
Surirella cf. crumena
Surirella ovalis
Surirella ovata
Surirella ovata var. apiculata
Surirella peisonis
Surirella striatula
Synedra famelica
Synedra radians
Synedra rumpens
Synedra ulna
Synedra ulna var. chaseana
Tabellaria flocculosa
Thalassiosira pseudonana
Thalassiosira sp.
Tryblionella apiculata
Tryblionella debilis
Tryblionella gracilis
Tryblionella hungarica
Tryblionella levidensis
Chlorarachnion globosum
Chlorarachnion reptans
Acetabularia acetabulum
Acetabularia caliculus
Acetabularia crenulata
Acetabularia dentata
Acetabularia farlowii
Acetabularia kilneri
Acetabularia major
Acetabularia ryukyuensis
Acicularia schenckii
Actinotaenium habeebense
Anadyomene stellata
Ankistrodesmus angustus
Ankistrodesmus arcuatus
Ankistrodesmus densus
Ankistrodesmus falcatus var. acicularis
Ankistrodesmus falcatus var. stipitatus
Ankistrodesmus nannoselene
Ankistrodesmus pseudobraunii
Ankistrodesmus sp.
Aphanochaete confervicola
Aphanochaete confervicola var. major
Aphanochaete elegans
Aphanochaete elegans var. minor
Arthrodesmus sp.
Ascochloris multinucleata
Asterococcus superbus
Astrephomene gubernaculifera
Atractomorpha echinata
Atractomorpha porcata
Axilococcus clingmanii
Axilosphaera vegetata
Basicladia sp.
Batophora occidentalis
Blastophysa rhizopus
Boergesenia forbesii
Boodlea composita
Boodlea montagnei
Bornetella oligospora
Bornetella sphaerica
Borodinellopsis texensis
Brachiomonas submarina
Brachiomonas submarina var. pulsifera
Bracteacoccus aerius
Bracteacoccus cohaerans
Bracteacoccus giganteus
Bracteacoccus grandis
Bracteacoccus medionucleatus
Bracteacoccus minor var. desertorum
Bracteacoccus minor var. glacialis
Bracteacoccus pseudominor
Bulbochaete hiloensis
Bulbochaete sp.
Capsosiphon fulvescens
Carteria crucifera
Carteria eugametos var. contaminans
Carteria olivieri
Carteria radiosa
Carteria sp.
Centrosphaera sp.
Cephaleuros parasiticus
Cephaleuros virescens
Chaetomorpha auricoma
Chaetomorpha spiralis
Chaetopeltis sp.
Chaetophora incrassata
Chaetosphaeridium globosum
Chalmasia antillana
Chamaetrichon capsulatum
Characiochloris acuminata
Characiosiphon rivularis
Characium acuminatum
Characium bulgariense
Characium californicum
Characium fusiforme
Characium hindakii
Characium oviforme
Characium perforatum
Characium polymorphum
Characium saccatum
Characium typicum
Chlamydomonas allensworthii
Chlamydomonas applanata
Chlamydomonas asymmetrica
Chlamydomonas callosa
Chlamydomonas chlamydogama
Chlamydomonas cribrum
Chlamydomonas culleus
Chlamydomonas debaryana var. cristata
Chlamydomonas desmidii
Chlamydomonas euryale
Chlamydomonas eustigma
Chlamydomonas fimbriata
Chlamydomonas gerloffii
Chlamydomonas gigantea
Chlamydomonas gloeophila var. irregularis
Chlamydomonas gyrus
Chlamydomonas hedleyi
Chlamydomonas hydra
Chlamydomonas inflexa
Chlamydomonas isabeliensis
Chlamydomonas leiostraca
Chlamydomonas lunata
Chlamydomonas melanospora
Chlamydomonas mexicana
Chlamydomonas minuta
Chlamydomonas minutissima
Chlamydomonas monadina
Chlamydomonas monoica
Chlamydomonas mutabilis
Chlamydomonas noctigama
Chlamydomonas oblonga
Chlamydomonas orbicularis
Chlamydomonas oviformis
Chlamydomonas perpusillus
Chlamydomonas philotes
Chlamydomonas proteus
Chlamydomonas provasolii
Chlamydomonas pseudagloe
Chlamydomonas pseudococcum
Chlamydomonas pulsatilla
Chlamydomonas pulvinata
Chlamydomonas pygmaea
Chlamydomonas radiata
Chlamydomonas rapa
Chlamydomonas sajao
Chlamydomonas simplex
Chlamydomonas smithii
Chlamydomonas sp.
Chlamydomonas sphaeroides
Chlamydomonas subangulosa
Chlamydomonas surtseyiensis
Chlamydomonas toveli
Chlamydomonas ulvaensis
Chlamydomonas yellowstonensis
Chlamydomonas zebra
Chlamydomonas zimbabwiensis
Chloranomala cuprecola
Chlorella anitrata
Chlorella anitrata var. minor
Chlorella antarctica
Chlorella ap.
Chlorella autotrophica var. atypica
Chlorella capsulata
Chlorella fusca var. fusca
Chlorella fusca var. vacuolata
Chlorella glucotropha
Chlorella luteoviridis
Chlorella miniata
Chlorella nocturna
Chlorella parva
Chlorella regularis var. minima
Chlorella saccharophila
Chlorella saccharophila var. saccharophila
Chlorella sp.
Chlorella sphaerica
Chlorella stigmatophora
Chlorella vulgaris
Chlorella zofingiensis
Chlorochytrium lemnae
Chlorocladus australasicus
Chlorococcales
Chlorococcum acidum
Chlorococcum aegyptiacum
Chlorococcum aquaticum
Chlorococcum arenosum
Chlorococcum citriforme
Chlorococcum croceum
Chlorococcum diplobionticum
Chlorococcum echinozygotum
Chlorococcum elbense
Chlorococcum elkhartiense
Chlorococcum gelatinosum
Chlorococcum granulosum
Chlorococcum isabeliense
Chlorococcum lacustre
Chlorococcum loculatum
Chlorococcum microstigmatum
Chlorococcum nivale
Chlorococcum novaeangliae
Chlorococcum oleofaciens
Chlorococcum oviforme
Chlorococcum paludosum
Chlorococcum pamirum
Chlorococcum perforatum
Chlorococcum perplexum
Chlorococcum pinguideum
Chlorococcum pulchrum
Chlorococcum pyrenoidosum
Chlorococcum refringens
Chlorococcum reticulatum
Chlorococcum rugosum
Chlorococcum salsugineum
Chlorococcum sphacosum
Chlorococcum tatrense
Chlorococcum texanum
Chlorococcum typicum
Chlorococcum uliginosum
Chlorocystis kornmannii
Chlorocystis westii
Chlorogonium perforatum
Chlorogonium sp.
Chlorogonium tetragamum
Chlorogonium tetragamum
Chloromonas actinochloris
Chloromonas asteroidea
Chloromonas augustae
Chloromonas brevispina
Chloromonas carrizoensis
Chloromonas chenangoensis
Chloromonas clathrata
Chlorosarcinopsis
Chlorosarcinopsis amylophila
Chlorosarcinopsis arenicola
Chlorosarcinopsis auxotrophica
Chlorosarcinopsis bastropiensis
Chlorosarcinopsis deficiens
Chlorosarcinopsis dissociata
Chlorosarcinopsis eremi
Chlorosarcinopsis halophila
Chlorosarcinopsis minor
Chlorosarcinopsis negevensis f. ferruguinea
Chlorosarcinopsis negevensis f. negevensis
Chlorosarcinopsis pseudominor
Chlorosarcinopsis sempervirens
Chlorosarcinopsis sp.
Chlorosarcinopsis variabilis
Coelastrum cambricum
Coelastrum proboscideum var. dilatatum
Coelastrum proboscideum var. gracile
Coelastrum sphaericum
Coenochloris planoconvexa
Cosmarium biretum
Cosmarium botrytis
Cosmarium connatum
Cosmarium cucumis
Cosmarium debaryi
Cosmarium formosulum
Cosmarium impressulum
Cosmarium margaritiferum
Cosmarium smolandicum
Cosmarium sp.
Cosmarium subcostatum
Cosmarium subtumidum
Cosmarium turpinii
Crucigenia lauterbornii
Crucigeniella rectangularis
Dictyococcus schumacherensis
Dictyococcus varians
Dictyosphaerium planctonicum
Diplostauron pentagonium
Gonium multicoccum
Gonium octonarium
Gonium quadratum
Gonium sacculiferum
Gonium sociale
Gonium sociale var. sacculum
Gonium sociale var. sociale
Gonium viridistellatum
Klebsormidium flaccidum var. cryophila
Klebsormidium marinum
Klebsormidium subtilissimum
Lagerheimia subsalsa
Mougeotia transeaui
Muriella aurantiaca
Muriella decolor
Mychonastes homosphaera
Nautococcus pyriformis
Nautococcus soluta
Neospongiococcum alabamense
Neospongiococcum butyrosum
Neospongiococcum commatiforme
Neospongiococcum concentricum
Neospongiococcum excentricum
Neospongiococcum giganticum
Neospongiococcum irregulare
Neospongiococcum macropyrenoidosum
Neospongiococcum mahleri
Neospongiococcum mobile
Neospongiococcum multinucleatum
Neospongiococcum proliferum
Neospongiococcum punctatum
Neospongiococcum rugosum
Neospongiococcum saccatum
Neospongiococcum solitarium
Neospongiococcum sphaericum
Neospongiococcum vacuolatum
Neospongiococcum variabile
Nephrochlamys subsolitaria
Oedogonium angustistomum
Oedogonium borisianum
Oedogonium calliandrum
Oedogonium cardiacum
Oedogonium donnellii
Oedogonium foveolatum
Oedogonium geniculatum
Oedogonium sp.
Oocystis alpina
Oocystis apiculata
Oocystis marssonii
Oocystis minuta
Oocystis sp
Pediastrum angulosum
Pediastrum boryanum var. cornutum
Pediastrum boryanum var. longicorne
Pediastrum clathratum
Pediastrum duplex var. asperum
Pediastrum simplex
Pediastrum sp.
Pithophora sp.
Pleurastrum erumpens
Pleurastrum terrestre
Pleurastrum terrestre var. indica
Protosiphon botryoides f. parieticola
Protosiphon sp.
Pseudendoclonium akinetum
Pseudendoclonium basiliensis
Pseudendoclonium prostratum
Pseudococcomyxa adhaerens
Raphidonema corcontica
Raphidonema longiseta
Raphidonema nivale
Raphidonema sp.
Raphidonema spiculiforme
Scenedesmus abundans
Scenedesmus arcuatus
Scenedesmus armatus
Scenedesmus basiliensis
Scenedesmus bijugatus var. seriatus
Scenedesmus breviaculeatus
Scenedesmus dispar
Scenedesmus hystrix
Scenedesmus jovais
Scenedesmus naegelii
Scenedesmus pannonicus
Scenedesmus parisiensis
Scenedesmus platydiscus
Scenedesmus sp.
Scenedesmus subspicatus
Selenastrum capricornutum
Selenastrum minutum
Selenastrum sp.
Sirogonium sticticum
Spirogyra condensata
Spirogyra crassispina
Spirogyra gracilis
Spirogyra grevilleana
Spirogyra juergensii
Spirogyra liana
Spirogyra maxima
Spirogyra meinningensis
Spirogyra notabilis
Spirogyra occidentalis
Spirogyra pratensis
Spirogyra quadrilaminata
Spirogyra rhizobrachialis
Spirogyra sp.
Spirogyra varians
Stichococcus & Heterococcus spp.
Stichococcus chodati
Stichococcus fragilis
Stichococcus mirabilis
Stichococcus sequoieti
Stigeoclonium aestivale
Stigeoclonium farctum
Stigeoclonium pascheri
Stigeoclonium subsecundum
Stigeoclonium tenue
Stigeoclonium variabile
Tetradesmus cumbricus
Zygnema amosum
Zygnema cylindricum
Zygnema extenue
Zygnema sp.
Zygnema spontaneum
Zygnema sterile
Campylomonas reflexa
Chroomonas coerulea
Chroomonas diplococca
Chroomonas pochmanii
Chroomonas sp.
Cryptochrysis sp.
Cryptomonas ovata
Cryptomonas ovata var. palustris
Cryptomonas ozolini
Cryptomonas sp.
Hemiselmis sp.
Proteomonas sulcata
Rhodomonas salina
Anabaena aequalis
Anabaena catenula
Anabaena cylindrica
Anabaena flos-aquae
Anabaena inaequalis
Anabaena minutissima
Anabaena randhawae
Anabaena sp.
Anabaena sphaerica
Anabaena spiroides
Anabaena subcylindrica
Anabaena subtropica
Anabaena variabilis
Anabaena verrucosa
Anacystis marina
Aphanizomenon flos-aquae
Arthrospira fusiformis
Calothrix anomala
Calothrix javanica
Calothrix membranacea
Calothrix parietina
Calothrix sp.
Chamaesiphon sp.
Chroococcidiopsis sp.
Cylidrospermum sp.
Cylindrospermopsis raciborskii
Cylindrospermum licheniforme
Cylindrospermum sp.
Dermocarpa sp.
Dermocarpa violacea
Entophysalis sp.
Eucapsis sp.
Fischerella ambigua
Fischerella muscicola
Fremyella diplosiphon
Gloeocapsa alpicola
Gloeocapsa sp.
Gloeotrichia echinulata
Gloeotrichia ghosi
Gloeotrichia sp.
Hapalosiphon welwitschii
Leptolyngbya nodulosa
Lyngbya aestuarii
Lyngbya kuetzingii
Lyngbya lagerheimii
Lyngbya purpurem
Lyngbya sp.
Mastigocladus laminosus
Merismopedia glauca f. insignis
Merismopedia sp.
Microcoleus sp.
Microcoleus vaginatus var. cyano-viridis
Microcystis aeruginosa
Microcystis flos-aquae
Microcystis sp.
Nodularia harveyana
Nodularia spumigena
Nostoc calcicola
Nostoc commune
Nostoc edaphicum
Nostoc ellipsosporum
Nostoc foliaceum
Nostoc longstaffi
Nostoc parmeloides
Nostoc piscinale
Nostoc punctiforme
Nostoc sp.
Nostoc zetterstedtii
Oscillatoria amoena
Oscillatoria animalis
Oscillatoria borneti
Oscillatoria brevis
Oscillatoria lud
Oscillatoria lutea
Oscillatoria lutea var. contorta
Oscillatoria prolifera
Oscillatoria sp.
Oscillatoria tenuis
Phormidium autumnale
Phormidium boneri
Phormidium foveolarum
Phormidium fragile
Phormidium inundatum
Phormidium luridum var. olivace
Phormidium persicinum
Phormidium sp.
Plectonema boryanum
Plectonema sp.
Pleurocapsa uliginosa
Porphyrosiphon notarisii
Rubidibacter lacunae
Schizothrix calcicola
Schizothrix calcicola var. radiata
Schizothrix calcicola var. vermiformis
Scytonema
Scytonema crispum
Scytonema hofmanni
Scytonema sp.
Spirirestis rafaelensis
Spirulina major
Spirulina maxima
Spirulina platensis
Spirulina sp.
Spirulina subsalsa
Spirulina subsalsa f. versicolor
Starria zimbabweensis
Symphyonemopsis katniensis
Symploca muscorum
Synechococcus
Synechococcus cedrorum
Synechococcus elongatus
Synechococcus sp.
Synechocystis nigrescens
Synechocystis sp.
Tolypothrix distorta var. symplocoides
Amphidinium carterae
Amphidinium rhynchocephalum
Ceratocorys horrida
Gyrodinium dorsum
Heterocapsa niei
Heterocapsa pygmeae
Karenia brevis
Oxyrrhis marina
Peridinium foliaceum
Peridinium inconspicuum
Peridinium sociale
Prorocentrum cassubicum
Prorocentrum triestinum
Pyrocystis lunula
Pyrocystis noctiluca
Scrippsiella trochoidea
Zooxanthella microadriatica
Colacium mucronatum
Colacium vesiculosum
Euglena acus var. gracilis
Euglena anabaena
Euglena cantabrica
Euglena caudata
Euglena deses
Euglena geniculata var. terricola
Euglena laciniata
Euglena mutabilis
Euglena myxocylindracea
Euglena pisciformis var. obtusa
Euglena proxima
Euglena rubra
Euglena sanguinea
Euglena sp.
Euglena spirogyra
Euglena stellata
Euglena terricola
Euglena tripteris
Eutreptia pertyi
Lepocinclis buetschlii
Lepocinclis ovata var. deflandriana
Phacus acuminata
Phacus brachykentron
Phacus caudata
Phacus megalopsis
Phacus pusillus
Phacus triqueter
Trachelomonas grandis
Trachelomonas hispida
Trachelomonas hispida var. coronata
Trachelomonas oblonga var. punctata
Trachelomonas volvocina
Trachelomonas volvocinopsis var. spiralis
Cyanophora biloba
Cyanophora paradoxa
Glaucocystis nostochinearum
Calyptrosphaera sphaeroidea
Chrysochromulina brevifilum
Coccolithophora sp.
Coccolithus neohelis
Cricosphaera carterae
Dicrateria inornata
Emiliania huxleyi
Isochrysis aff. galbana
Isochrysis galbana
Isochrysis sp.
Ochrosphaera neapolitana
Ochrosphaera verrucosa
Pavlova gyrans
Pavlova lutheri
Pseudoisochrysis paradoxa
Sarcinochrysis marina
Asterosiphon dichotomus
Aureoumbra lagunensis
Bodanella lauterborni
Botrydiopsis arhiza
Botrydium cystosum
Bumilleria exilis
Bumilleria sicula
Bumilleriopsis sp.
Chattonella japonica
Chloridella miniata
Chlorocloster solani
Chlorocloster sp.
Chromulina nebulosa
Chrysochaete britannica
Dictyopteris repens
Dictyota cilliolata
Dictyota dichotoma
Dinobryon sp.
Ectocarpus siliculosus
Ectocarpus sp.
Ectocarpus variabilis
Ellipsoidion sp.
Epipyxis pulchra
Eustigmatos magna
Heterococcus caespitosus
Heterococcus cf. caespitosus
Heterococcus cf. endolithicus
Heterococcus cf. pleurococcoides
Heterococcus cf. protnematoides
Heterococcus chodati
Heterococcus fuornensis
Heterococcus mainxii
Heterococcus moniliformis
Heterococcus protonematoides
Heterococcus sp.
Heterococcus sp. Pleuroscoccoides
Heterothrix debilis
Heterotrichella gracilis
Hibberdia magna
Lagynion scherffelii
Mallomonas asmundae
Mischococcus sphaerocephalus
Monodus subterraneus
Nannochloropsis oculata
Ochromonas sp.
Ochromonas spherocystis
Ophiocytium maius
Phaeoplaca thallosa
Phaeoschizochlamys mucosa
Pleurochloris meiringensis
Pseudobumilleriopsis pyrenoidosa
Sorocarpus uvaeformis
Spermatochnus paradoxus
Sphacelaria cirrosa
Sphacelaria rigidula
Sphacelaria sp.
Stichogloea doederleinii
Synura petersenii
Synura uvella
Tribonema missouriense
Tribonema sp.
Vacuolaria virescens
Vaucheria bursata
Vaucheria geminata
Vaucheria sessilis
Vaucheria terrestris
Vischeria punctata
Acrochaetium flexuosum
Acrochaetium pectinatum
Acrochaetium plumosum
Acrochaetium proskaueri
Acrochaetium sagraeanum
Acrochaetium sp
Acrosorium uncinatum
Anfractutofilum umbracolens
Antithamnion defectum
Antithamnion glanduliferum
Apoglossum ruscifolium
Asterocytis ramosa
Asterocytis sp.
Audouinella eugenea
Audouinella hermannii
Bangia afusco-purpure
Bangia atro-purpurea
Bangia fusco-purpurea
Bangiopsis subsimplex
Batrachospermum intortum
Batrachospermum macrosporum
Batrachospermum moniliforme
Batrachospermum sirodotia
Batrachospermum sp.
Batrachospermum vagum var. keratophylum
Boldia erythrosiphon
Bostrychia bispora
Bostrychia tenella
Botryocladia ardreana
Botryocladia boergesenii
Botryocladia pyriformis
Bryothamnion triqutrum
Callithamnion baileyi
Callithamnion byssoides
Callithamnion corymbosum
Callithamnion halliae
Callithamnion paschale
Callithamnion roseum
Callithamnion sp.
Caloglossa intermedia
Caloglossa leprieurii f. pygmaea
Ceramium sp.
Champia parvula
Chondrus crispus
Compsopogon coeruleus
Compsopogon hookeri
Compsopogon oishii
Compsopogonopsis leptoclados
Cumagloia andersonii
Cyanidium caldarium
Cystoclonium purpureum
Dasya pedicellata
Dasya rigidula
Digenea simplex
Dixoniella grisea
Erythrocladia sp.
Erythrotrichia carnea
Eupogodon planus
Flintiella sanguinaria
Gelidiopsis intricata
Glaucosphaera vacuolata
Gracilaria debilis
Gracilaria foliifera
Gracilaria verrucosa
Grateloupia filicina
Griffithsia pacifica
Heterosiphonia plumosa
Hildenbrandia prototypus
Hildenbrandia rivularis
Hypnea musciformis
Lomentaria articulata
Lomentaria orcadensis
Lophocladia trichoclados
Nemalion multifidum
Nemalionopsis shawi f. caroliniana
Nemalionopsis tortuosa
Neoagardhiella baileyi
Palmaria palmata
Phyllophora membranacea
Phyllophora truncata
Polyneura hilliae
Polyneura latissima
Polysiphonia boldii
Polysiphonia echinata
Porphyra eucosticta
Pseudochantransia sp.
Pterocladia americana
Pterocladia bartlettii
Pterocladia capillacea
Ptilothamnion sp.
Purpureofilum apyrenoidigerum
Rhodella maculata
Rhodochaete parvula
Rhodochorton purpureum
Rhodochorton tenue
Rhodosorus marinus
Rhodospora sordida
Rhodymenia cf. Ardisonnei Rard Cor
Rhodymenia pseudopalmata
Seirospora griffithsiana
Sirodotia sp.
Sirodotia suecica
Sirodotia tenuissima
Solieria tenera
Spermothamnion speluncarum
Spermothamnion turneri
Spyridia filimentosa
Stylonema alsidii
Thorea hispida
Thorea okaida
Thorea riekei
Thorea violacea
Trailliella intricata
Tuomeya americana
Tuomeya fluviatilis
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
radiosa
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
rubescens
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
Desmodesmus
armatus
Desmodesmus
brasiliensis
Desmodesmus cf. corallinus
Desmodesmus cf. gutwinskii
Desmodesmus cf. opoliensis var. mononensis
Desmodesmus cf. pannonicus
Desmodesmus cf. spinosus
Desmodesmus
fuscus
Desmodesmus
granulatus
Desmodesmus
hirsutus
Desmodesmus
quadricauda
Desmodesmus
sempervirens
Desmodesmus
subspicatus
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.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2009/050202 | 7/10/2009 | WO | 00 | 8/31/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61080031 | Jul 2008 | US | |
| 61087077 | Aug 2008 | US |
