CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/350,898, filed June 2, 2010, which application is herein incorporated by reference in its entirety.

BACKGROUND

[0002] Oil spills have unwanted effects to the environment. Such spills are generally treated through physical containment of the spilled oil and removal, using mechanical techniques, such as containment rings and vacuum removal systems. Other means for treating such spills include direct application to the spill of dispersants and application of bioremediation agents, such as aerobic micro-organisms, enzymes and nutrients. Since these reagents are often not native to environment of the oil spill, they may in fact have toxic effects to the environment in which they are delivered. Limited success in adequately dealing with such spills evidences the need for a product and method for dealing with an oil spill promptly, irrespective of its location, configuration and accessibility, and with minimum disruption to the environment.

INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

SUMMARY

[0004] In one aspect, disclosed herein is a method of evolving a microorganism comprising (a) artificially evolving a microorganism by continuously culturing the microorganism in a first medium comprising a complex hydrocarbon, (b) selecting a microorganism robustly growing in the first medium, and (c) inoculating the robustly growing microorganism into a second medium comprising higher percentage of complex hydrocarbon than that of the first medium. In one embodiment, the complex hydrocarbon is a hydrocarbon molecule found in crude oil, petroleum, Texas light sweet, Louisiana sweet, Brent crude, Pennsylvania grade crude oil, Sour crude oil, Dubai crude, Mazut crude or light crude oil. In another embodiment, the microorganism is Alcanivorax borkumensis, Alcanivorax dieselolei, Alcanivorax jadensis, Azoarcus toluvorans, Desulfatibacillum aliphaticivorans, Desulfotomaculum sp., Gordonia alkanivorans, Marinobacter hydrocarbonoclasticus, Neptunomonas naphthovorans, Novosphingobium aromaticivorans, Rhodococcus erythropolis, Sphingobium yanoikuyae, Sphingomonas sp.,

Tranquillimonas alkanivorans, Tropicimonas isoalkanivorans, Thauera aromatica, or Thalassolituus oleovorans. In another embodiment, one part of the first medium is a complex hydrocarbon and nine parts of the first medium is microbial medium. In another embodiment, the selecting is visually inspecting the culture for turbidity. In another embodiment, the higher percentage is selected from the group consisting of two, three, four, five, six, seven, eight, or nine parts of a complex hydrocarbon. In another embodiment, the first medium and/or the second medium further comprises seawater. In another embodiment, the continuous culture is maintained at 28 °C. In another embodiment, the robustly growing microorganism grows faster and/or to a higher cell density than before the artificial evolution.

[0005] In another aspect, disclosed herein is a method of evolving a microorganism comprising (a) artificially evolving a microorganism by continuously culturing the microorganism in a first medium comprising a complex hydrocarbon and a dispersant, (b) selecting a microorganism robustly growing in the first medium, and (c) inoculating the robustly growing microorganism into a second medium comprising higher percentage of complex hydrocarbon than that of the first medium. In one embodiment, the complex hydrocarbon is a hydrocarbon molecule found in crude oil, petroleum, Texas light sweet, Louisiana sweet, Brent crude, Pennsylvania grade crude oil, Sour crude oil, Dubai crude, Mazut crude or light crude oil. In another embodiment, the microorganism is Alcanivorax borkumensis, Alcanivorax dieselolei, Alcanivorax jadensis, Azoarcus toluvorans, Desulfatibacillum aliphaticivorans,

Desulfotomaculum sp., Gordonia alkanivorans, Marinobacter hydrocarbonoclasticus, Neptunomonas naphthovorans, Novosphingobium aromaticivorans, Rhodococcus erythropolis, Sphingobium yanoikuyae, Sphingomonas sp., Tranquillimonas alkanivorans, Tropicimonas isoalkanivorans, Thauera aromatica, or Thalassolituus oleovorans. In another embodiment, one part of the first medium is a complex hydrocarbon and nine parts of the first medium is microbial medium. In another embodiment, the selecting is visually inspecting the culture for turbidity. In another embodiment, the higher percentage is selected from the group consisting of two, three, four, five, six, seven, eight, or nine parts of a complex hydrocarbon. In another embodiment, the first medium and/or the second medium further comprises seawater. In another embodiment, the continuous culture is maintained at 28 °C. In another embodiment, the dispersant contains 2-butoxyethanol. In another embodiment, the robustly growing microorganism grows faster and/or to a higher cell density than before the artificial evolution.

[0006] In another aspect, disclosed herein is a composition comprising (a) an artificially evolved microorganism, (b) petroleum, (c) a dispersant, and (d) water. In one embodiment, the water is seawater. In another embodiment, the the dispersant contains 2-butoxyethanol. In another embodiment, the composition further comprising a plurality of artificially evolved microorganisms wherein the plurality of artificially evolved microorganisms comprises a community of microbial strains.

[0007] In another aspect, disclosed herein is a method of evolving a microorganism comprising (a) artificially evolving a microorganism by continuously culturing the microorganism in a first medium comprising petroleum, (b) selecting a microorganism robustly growing in the medium, and (c) inoculating the robustly growing microorganism into a second medium comprising higher percentage of petroleum than that of the first medium.

[0008] In another aspect, disclosed herein is a method of evolving a microorganism comprising (a) artificially evolving a microorganism by continuously culturing the microorganism in a first medium comprising petroleum and a dispersant, (b) selecting a microorganism robustly growing in the medium, and (c) inoculating the robustly growing microorganism into a second medium comprising higher percentage of petroleum than that of the first medium.

[0009] In another aspect, disclosed herein is a method of bioremediation comprising (a) applying to an environment contaminated with a complex hydrocarbon an evolutionarily modified organism (EMO), wherein the EMO has been artificially evolved to break down the complex hydrocarbon, and (b) providing sufficient time for the EMO to break down the complex hydrocarbon, wherein the EMO breaks down the complex hydrocarbon faster or more efficiently than a non-evolutionarily modified organism. In one embodiment, the environment is a beach, wetlands, river bottom, mudflats, stream, river, sea, or ocean. In another embodiment, a dispersant has been administered to the complex hydrocarbon. In another embodiment, the complex hydrocarbon is petroleum. In another embodiment, the EMO breaks down the complex hydrocarbon 1 to 2 times faster than a non-evolutionarily modified organism. In another embodiment, the EMO breaks down the complex hydrocarbon 3 to 4 times faster than a non- evolutionarily modified organism. In another embodiment, the EMO breaks down the complex hydrocarbon more than 4 times faster than a non-evolutionarily modified organism.

[0010] In another aspect, disclosed herein is a method of bioremediation comprising (a) applying to an environment contaminated with petroleum an evolutionarily modified organism (EMO), wherein the EMO has been artificially evolved to break down the petroleum, and (b) providing sufficient time for the EMO to break down the petroleum, wherein the EMO breaks down the petroleum faster or more efficiently than a non-evolutionarily modified organism. In one embodiment, a dispersant has been administered to the petroleum. In another embodiment, the EMO breaks down the petroleum 1 to 2 times faster than a non-evolutionarily modified organism. In another embodiment, the EMO breaks down the petroleum 3 to 4 times faster than a non-evolutionarily modified organism. In another embodiment, the EMO breaks down the petroleum more than 4 times faster than a non-evolutionarily modified organism.

[0011] In another aspect, disclosed herein is a method of bioremediation comprising (a) applying to an environment contaminated with a complex hydrocarbon an evolutionarily modified organism (EMO), wherein the EMO is the evolved microorganism produced by the method of claims 1 or 2, and (b) providing sufficient time for the EMO to break down the complex hydrocarbon, wherein the EMO breaks down the complex hydrocarbon faster or more efficiently than a non- evolutionarily modified organism. In one embodiment, the complex hydrocarbon is petroleum. In another embodiment, the EMO breaks down the complex hydrocarbon 1 to 2 times faster than a non-evolutionarily modified organism. In another embodiment, the EMO breaks down the complex hydrocarbon 3 to 4 times faster than a non- evolutionarily modified organism. In another embodiment, the EMO breaks down the complex hydrocarbon more than 4 times faster than a non-evolutionarily modified organism. INCORPORATION BY REFERENCE

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0014] Figure 1 illustrates petroleum spill taken from a seawater spill (A) or a spill containing dispersant (B).

[0015] Figure 2 (FIGs. 2A and 2B) illustrates improved growth of adapted community of microbial strains on oil as a sole substrate.

[0016] Figure 3 illustrates improved growth rate of adapted community of microbial strains adapted for 3 weeks or for 5 weeks.

[0017] Figure 4 illustrates an overall view of a possible configuration of the device in which: (1) represents the flexible tubing containing the different regions of the device which are: upstream fresh medium (7), growth chamber (10), sampling chamber (11) and disposed grown culture region (15). (2) represents the thermostatically controlled box allowing regulation of temperature according to conditions determined by user, and in which may be located: a. said growth chamber (10), b. said sampling chamber (11), c. upstream gate (3) defining the beginning of said growth chamber (10), d. downstream gate (4) defining the end of said growth chamber (10) and the beginning of said sampling chamber (11), e. second downstream gate (5) defining the end of said sampling chamber (11), f. turbidimeter (6) allowing the user or automated control system to monitor optical density of growing culture and to operate a feedback control system (13), allowing controlled movement of the tubing (1) on the basis of culture density (turbidostat function), and g. one or several agitators (9). It should be noted that the device elements listed in a-g may also be located outside of, or in the absence of, a thermostatically controlled box. (7) represents the fresh medium in unused flexible tubing. (8) represents a barrel loaded with fresh medium filled tubing, in order to dispense said fresh medium and tubing during operations. (12) represents optional ultra-violet radiation gates. (13) represents the control system that can consist of a computer connected with means of communication to different monitoring or operating interfaces, like optical density turbidimeters, temperature measurement and regulation devices, agitators and tilting motors, etc, that allow automation and control of operations, (14) represents the optional disposal barrel on which to wind up tubing containing disposed grown culture filled tubing, (15) represents disposed grown culture located downstream of said sampling chamber.

DETAILED DESCRIPTION

[0018] The biodegradation rate of wild-type oil-degrading microorganism is relatively slow to be useful as a bioremediation agent to treat large-scale petroleum spill or to cover large amount of continuous influx of petroleum products, such as a landfill. It has been postulated that adaptation to a particular petroleum- rich environment, which is different to the microorganism's natural flora, may have been a contributing factor. A sudden, large amount or percentage of petroleum products would understandably bring a challenge to a microorganism to cope with the change in nutrient and oxygen environment. Activation of metabolic pathways designed to metabolize certain hydrocarbon or nitrogen sources in petroleum products can take time, which also adds a total time to reach robust biodegradation of petroleum products.

[0019] Disclosed herein are compositions and methods of evolving microorganisms to adapt oil- degrading marine oligotrophs for high concentrations of oil and improved growth rates. Methods disclosed herein are applicable to a variety of known oil-degraders that specialize in metabolizing compounds from different chemical classes (aliphatics, aromatics, polyaromatic hydrocarbons, asphaltenes) to adopt improved biodegradation properties allowing the application of evolved

microorganism s an industrially feasible reality. An improved biodegradation property includes not only increased growth rate or biodegradation rate, but also increased tolerance to dispersant, other chemicals, or physical parameters (such as pH, salinity, pressure, temperature) that can inhibit the growth of a microorganism. The content of crude oil varies widely depending on the source of the oil. Methods disclosed herein can artificially evolve a microorganism to grow on a particular type of crude oil. In addition, methods disclosed herein can artificially evolve a microorganism to grow on a particular type of crude oil spilled in a particular environment, e.g., sea, lake, river, landfill, mud, sand, groundwater, or wetland. Methods disclosed herein also provide for evolving a microorganism in which its rapid growth rate depends on oil and thus the population of microorganism is expected to decline significantly as the availability of oil is diminished.

[0020] Provided herein are compositions and methods for growing an evolutionarily modified organism (EMO) on a medium comprising petroleum or petroleum products comprising one or more complex hydrocarbons. An EMO described herein is useful for biodegrading petroleum or petroleum products comprising one or more complex hydrocarbons. An EMO described herein can be provided as an environmentally friendly alternative, or as an additional remedy to other known methods of petroleum containment. For example, an EMO described herein is useful to biodegrade petroleum in seawater in the presence or absence of a dispersant.

[0021] There are a variety of naturally occurring microbes capable of metabolizing hydrocarbons as the sole source of carbon or energy. Bioremediation is a process to harness the degradative power of these microbes to accelerate recovery from oil spills of anthropogenic origin. In the past, bioremediation has consisted of two main approaches: biostimulation and bioaugmentation.

[0022] Biostimulation is premised on the concept that after an oil spill, the natural oil-degrading flora blooms, but their growth rate is limited by low levels of other nutrients essential for robust growth and expansion of natural, oil-degrading microbes. To remedy this problem, affected areas are fertilized with nutrients to stimulate the growth of indigenous oil-eating microbes. Biostimulation has certain limitations. First, marine environment have been shown to contain sufficient nutrient levels, suggesting that nutrient acquisition may not be a limiting factor for biodegradation. In certain environment, such as the Gulf of Mexico, oxygen deficiency seems to be more of a limiting factor than nutrient deficiency. Second, oil forms a separate phase from water, making access to the substrate another critical limiting factor for microbial growth. Third, any nutrients that are added to seawater biostimulate both oil-eating and non-oil degraders. None oil degraders, some of which may even be pathogens, can outcompete the growth rate of oil-eating microbes if they preferentially digest added nutrients. Lastly, the interfering effect of chemical dispersants to natural oil-eating microbes is not known.

[0023] Bioaugmentation is the introduction of a group of natural microbial strains or a genetically engineered variant to treat contaminated soil or water. Crude oil consists of five general classes of hydrocarbons: n-alkanes (paraffins), cycloalkanes (naphthenes), aromatics, polycyclic aromatics (PAHs) and asphaltics. The amount and diversity of compounds in each group depends heavily on the source of the crude oil. In bioaugmentation, affected areas are seeded with microbes that are specialized for the biodegradation of particular contaminants that are refractory to decomposition by native flora. A diverse array of microbes has been isolated for their capability of degrading individual components of crude oil. Bioaugmentation can be limited depending on the environment in which the group of microbes are introduced because as non-indigenous microbes, they may not compete well with autochthonous microbes.

[0024] Described herein are methods and compositions in which a community of microbial strains

(CMS) is evolutionarily adapted to grow robustly in a non- indigenous environment. In one embodiment, a CMS is evolutionarily adapted by a continuous culture system described herein. In another embodiment, a CMS is evolutionarily modified by a system described in U.S. Patent Application Publication No.

20070037276 and U.S. Patent No. 7,939,315, both of which are herein incorporated by refereeing each in their entirety. In one embodiment, a CMS is formed by selecting microbes for their ability to digest oil in a particular environmental condition. In one embodiment, a CMS is modified to a particular seawater condition. In one embodiment, the evolutionarily modified community of microbial strains (EMCMS) is utilized to clean up oil spill in seawater.

Definitions:

[0025] As used herein, a "dispersant" is a chemical that aids in breaking up solids or liquids as fine particles or droplets into another medium. A dispersant includes emulsifiers of oil into water or water into oil, surfactants, such as those selected on the basis of Hydrophilic-lipophilic balance (HLB) number including cocoamide, synthetic detergents, soaps, organic solvent such as 2-butoxyethanol, or other polymers such as propylene glycol.

[0026] As used herein, "complex hydrocarbon" refers to one or more hydrocarbon molecules found in crude oil, petroleum, sweet crude such as Texas light sweet, Louisiana Sweet, Brent Crude, Pennsylvania Grade Crude oil, Sour crude oil, Dubai Crude, Mazut, Light Crude oil, in underground oil reservoir or in any petroleum products thereof. Non- limiting examples of hydrocarbon molecules include alkanes, aklenes, alkyenes, cyclolakanes, aromatic hydrocarbons, asphaltics, and other organic molecules containing nitrogen, oxygen and/or sulfur. Alkanes include C5 hydrocarbon to C 16 hydrocarbon. Alkanes include paraffins. Alkenes include hydrocarbon molecules with a general formula of C _n H2 _n wherein n equals to a whole number 1 to 20. Alkyenes include hydrocarbon molecules with a general formula of C _n H2 _n -2 wherein n equals to a whole number 1 to 20. Cycloalkane or naphthenes include hydrocarbon molecules containing a ring structure with a general formula of CnH2n wherein n equals to a whole number 1 to 20, such as cyclohexane or methyl cyclopentane. Aromatic hydrocarbons include ring- containing hydrocarbon molecules such as benzene or naphthalene. A complex hydrocarbon can include methylene chloride, 1, 1-dichloroethane, chloroform, 1,2-dichloropropane, dibromochloromethane, 1, 1,2- trichloroethane, 2-chloroethylvinyl ether, tetrachloroethene (PCE), chlorobenzene, 1,2-dichloroethane, 1, 1, 1-trichloroethane, bromodichloromethane, trans- 1,3-dichloropropene, cis- l,3-dichloropropene, bromoform, chloromethane, bromomethane, vinyl chloride, chloroethane, 1, 1-dichloroethene, trans- 1,2- dichloroethene, trichloroethene (TCE), dichlorobenzenes, cis- l,2-dichloroethene, dibromomethane, 1,4- dichlorobutane, 1,2,3-trichloropropane, bromochloromethane, 2,2-dichloropropane, 1,2-dibromoethane, 1,3-dichloropropane, bromobenzene, chlorotoluenes, trichlorobenzenes, trans- l,4-dichloro-2-butene and butylbenzenes.

[0027] The terms "carbonaceous material" or "biomass" as used herein includes a biological materials that can be converted into a biofuel, chemical or other end product. One exemplary source of carbonaceous material is an_agricultural product. One exemplary source of carbonaceous material is plant matter. Plant matter can be, for example, woody plant matter, non- woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, sugar cane, grasses, switchgrass, bamboo, and material derived from these. Plant matter can also be residual spent solids from alcoholic fermentation from materials such as corn and which contain lignin, starch, cellulose, hemicellulose, and proteins. Plant matter can be further described by reference to the chemical species present, such as proteins, polysaccharides (such as chitin) and oils. Polysaccharides include polymers of various monosaccharides and derivatives of monosaccharides including glucose, fructose, lactose, galacturonic acid, rhamnose, etc. Plant matter also includes agricultural waste byproducts or side streams such as pomace, corn steep liquor, corn steep solids, corn stover, corn stillage, corn cobs, corn grain, bagasse, soy stems, soy leaves, soy pods, soy molasses , soy flakes, pennycress seeds or seed cake, distillers grains, peels, pits, fermentation waste, wood chips, saw dust, wood flour, wood pulp, paper pulp, paper pulp waste steams straw, lumber, sewage, seed cake, husks, rice hulls, leaves, grass clippings, food waste restaurant waste, or cooking oil. These materials can come from farms, forestry, industrial sources, households, etc. Plant matter also includes maltose, corn syrup, Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), or Distillers Dried Grains with Solubles (DDGS). Biomass includes animal matter such as milk, meat, fat, bone meal, animal processing waste, and animal waste (e.g., feces). "Feedstock" is frequently used to refer to biomass being used for a process, such as those described herein. Another example of carbonaceous material or biomass is glycerol (such as unpurified glycerol from a transesterification process), sewage, municipal waste, which can contain indigestible materials such as paper or other cellulosic, hemicellulosic or lignocellulosic material.

[0028] The term "fatty acid" or lipid or oil as used herein includes one or more chemical compounds that include one or more fatty acid moieties as well as derivatives of these compounds and materials that comprise one or more of these compounds. Common examples of compounds that include one or more fatty acid moieties include triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids, lysophospholipids, free fatty acids, fatty acid salts, soaps, fatty acid comprising amides, esters of fatty acids and monohydric alcohols, esters of fatty acids and polyhydric alcohols including glycols (e.g., ethylene glycol, propylene glycol, etc.), esters of fatty acids and polyethylene glycol, esters of fatty acids and polyethers, esters of fatty acids and polyglycol, esters of fatty acids and saccharides, esters of fatty acids with other hydroxyl-containing compounds, etc. A "fatty acid" can include one or more volatile fatty acids such as acetic acid, propionic acid, butyric acid, butyrate, caproate, caprylate, valerate and heptanoate, and the like. "Fatty acids" include saturated and unsaturated fatty acids. The fatty acid portion of the fatty acid comprising compound can be a simple fatty acid, such as one that includes a carboxyl group attached to a substituted or un-substituted alkyl group. The substituted or unsubstituted alkyl group can be straight or branched, saturated or unsaturated. Substitutions on the alkyl group can include hydroxyls, phosphates, halogens, alkoxy, or aryl groups. The substituted or unsubstituted alkyl group can have 7 to 30 carbons, such as 11 to 23 carbons (e.g., 8 to 30 carbons or 12 to 24 carbons counting the carboxyl group) arranged in a linear chain with or without side chains and/or substitutions. A fatty acid comprising material can be one or more of these compounds in an isolated or purified form. It can be a material that includes one or more of these compounds that is combined or blended with other similar or different materials.

[0029] As used herein, the terms "petroleum pollutants" or "petroleum products" include compounds contained in crude or refined petroleum. Examples of such compounds include without limitation Texas light sweet, Louisiana Sweet, Brent Crude, Pennsylvania Grade Crude oil, Sour crude oil, Dubai Crude, Mazut, and Light Crude oil. Adddtionally, such petroleum-derived compounds typically include combinations of aliphatics (e.g., C5-C36) and aromatics (e.g., C9-C22). Specific petroleum pollutants include crude oil, refined oil, fuel oils (e.g., Nos. 2, 4 and 6 fuel oils), diesel oils, gasoline, hydraulic oils and kerosene. Benzene, toluene, ethylbenzene and xylenes (BTEX) are the most volatile constituents of gasoline and may be present in the petroleum pollutants. Trimethylbenzenes, and other PAHs such as naphthalene, anthracene, acenaphthene, acenaphthylene, benzo (a) anthracene, benzo (a) pyrene, benzo (b) fluoranthene, benzo (g,h,i) perylene, benzo (k) fluoranthene and pyrene, are also common constituents of fuel oils and heavier petroleum compounds. The amount of petroleum pollutants contained in a contaminated site, for example in soil or groundwater, may be quantified as total petroleum hydrocarbons (TPH). In addition, the petroleum pollutants may be quantified as individual groups of aliphatic and aromatic compounds.

[0030] As used herein, the term "community" refers to a collection of microbial strains pooled together.

Evolutionary Modification of An Organism

[0031] Provided herein is an EMO adapted to grow on a medium comprising complex hydrocarbon. An EMO is an organism that has been artificially evolutionarily modified by culturing the organism for a sufficient period of time under controlled conditions to induce one or more changes in the organism's DNA (e.g., a point mutation, a translocation, missense mutation, a nonsense mutation, duplication, deletion or gene rearrangement, methylation), or to induce one or more changes to the organism's chromatin (e.g., histone methylation or acetylation) that causes a desired phenotypic change. In one embodiment an EMO has been modified so that it contains multiple mutations that effect one or more genes. In another embodiment an EMO has been modified by continuous culture. In one embodiment an EMO is a single celled organism. In another embodiment an EMO is a microorganism. In another embodiment an EMO is a bacterium. In another embodiment an EMO is an alga. In another embodiment an EMO is a multicellular organism.

[0032] In one embodiment, an EMO is adapted to grow on a medium comprising petroleum or petroleum products. An example of petroleum includes, but is not limited to, crude oil, petroleum, sweet crude such as Texas light sweet, Louisiana Sweet, Brent Crude, Pennsylvania Grade Crude oil, Sour crude oil, Dubai Crude, Mazut, Light Crude oil, or in underground oil reservoir. In another embodiment, the EMO is adapted to grow on a medium comprising petroleum and water. In one embodiment the water is fresh water. In another embodiment the water is salt water. In another embodiment, the EMO is adapted to grow on a medium comprising petroleum and seawater. In another embodiment, the EMO is adapted to grow on a medium comprising petroleum, mud and water. In another embodiment, the EMO is adapted to grow on a medium comprising gasoline. In another embodiment, the EMO is adapted to grow on a medium comprising diesel fuel. In another embodiment, the EMO is adapted to grow on a medium comprising one or more complex hydrocarbons. In another embodiment, the EMO is adapted to grow on a medium comprising one or more naphthenes. In another embodiment, the EMO is adapted to grow on a medium comprising one or more asphaltenes. In another embodiment, the EMO is adapted to grow on a medium comprising one or more complex hydrocarbons and another EMO. In another embodiment, the EMO is adapted to grow on a medium comprising one or more complex hydrocarbons and one or more dispersants. In one embodiment, a dispersant useful for adapting an EMO for tolerance is 2- butoxyethanol. In another embodiment, a dispersant useful for adapting an EMO for tolerance is polyoxyethylene sorbitol hexaoleate. In another embodiment, a dispersant useful for adapting an EMO for tolerance is ethylene oxide. In another embodiment, a dispersant useful for adapting an EMO for tolerance is isopropylamine dodecyl benzene sulphonate. In another embodiment, a dispersant useful for adapting an EMO for tolerance is sodium dodecyl sulfate. In another embodiment, a dispersant useful for adapting an EMO for tolerance is ethylene glycol monobutyl ether. In another embodiment, a dispersant useful for adapting an EMO for tolerance is butyl oxitol. In another embodiment, a dispersant useful for adapting an EMO for tolerance is polyexyethylene sorbitol hexaoleate. In another embodiment, a dispersant useful for adapting an EMO for tolerance is calcium dodecyl benzene sulphonate. In another embodiment, a dispersant useful for adapting an EMO for tolerance is polyoxyethylene sorbitan trioleate.

[0033] In one embodiment, an EMO is evolved to adapt to a low oxygen environment by continuously culturing an EMO in a hypoxic condition. In one embodiment a hypoxic condition comprises between 1- 30% oxygen. In one embodiment, a hypoxic condition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 percent oxygen. In one embodiment the EMO is further cultured with a complex hydrocarbon, such as petroleum. In another embodiment, an EMO is evolved to adapt to a low oxygen, low nitrogen and/ or low phosphorous environment by continuously culturing an EMO in a hypoxic condition low nitrogen and/or low phosphorous conditions. In one embodiment the continuously culturing condition comprises decreasing an amount of nitrogen provided from a microbial culture medium, such as yeast extract.

[0034] In one embodiment, an EMO is evolved to adapt to an low oxygen environment by continuously culturing an EMO in anoxic conditions. In one embodiment an anoxic condition comprises less than 1% oxygen. In one embodiment the EMO is further cultured with a complex hydrocarbon, such as petroleum. In another embodiment, an EMO is evolved to adapt to a low oxygen, low nitrogen and/ or low phosphorous environment by continuously culturing an EMO in anoxic conditions, low nitrogen and/or low phosphorous conditions. In one embodiment the EMO is further cultured with a complex hydrocarbon, such as petroleum. In one embodiment the continuously culturing condition comprises decreasing an amount of nitrogen provided from a microbial culture medium, such as yeast extract.

[0035] In one embodiment, an EMO is evolved to adapt to a low oxygen environment by continuously culturing an EMO in anaerobic conditions. In one embodiment the EMO is further cultured with a complex hydrocarbon, such as petroleum. In another embodiment, an EMO is evolved to adapt to allow oxygen, low nitrogen and/ or low phosphorous environment by continuously culturing an EMO under anaerobic conditions, low nitrogen and/or low phosphorous conditions. In one embodiment the EMO is further cultured with a complex hydrocarbon, such as petroleum. In one embodiment the continuously culturing condition comprises decreasing an amount of nitrogen provided from a microbial culture medium, such as yeast extract.

[0036] In one embodiment, an EMO is evolved to adapt to a environment by continuously culturing an EMO in aerobic conditions. In one embodiment a aerobic condition comprises about 80% or more oxygen. In one embodiment the EMO is further cultured with a complex hydrocarbon, such as petroleum. In another embodiment, an EMO is evolved to adapt to a low nitrogen and/ or low phosphorous environment by continuously culturing an EMO under aerobic conditions, low nitrogen and/or low phosphorous conditions. In one embodiment the EMO is further cultured with a complex hydrocarbon, such as petroleum. In one embodiment the continuously culturing condition comprises decreasing an amount of nitrogen provided from a microbial culture medium, such as yeast extract.

[0037] In one embodiment, an EMO is evolved to adapt to a high salinity environment by continuously culturing an EMO in the prescience of high salt concentrations. In one embodiment high salt

concentrations comprises 33% more salt than normal saline conditions. In one embodiment the EMO is further cultured with a complex hydrocarbon, such as petroleum.

[0038] In one embodiment, an EMO is evolved to adapt to a normal salinity environment by continuously culturing an EMO in the prescience of normal salt concentrations. In one embodiment the EMO is further cultured with a complex hydrocarbon, such as petroleum.

[0039] The EMO can be produced by a method, device, or composition as described herein. In one embodiment, the evolutionary modification process uses a continuous culture method described in U.S. Patent Applications 20070037276 or U.S. Patent Publication 7,939,315 which is herein incorporated by reference in their entirety. In one embodiment an organism is evolutionarily modified with an

Evolugator™ (e.g., FIG. 4) to produce an EMO specifically modified to have a desired phenotype. In another embodiment the phenotype is increased growth rate, increased production of a end product (e.g., fatty acids or alcohol), increased culture density, or decreased production of an end-product (e.g., acetate, lactate, H ₂ or C0 ₂ ).

[0040] The evolutionary process described herein can be applied to a wide range of organisms. In one embodiment, an organism evolves to acquire one or more industrially useful characteristics. In another embodiment, an organism is evolved to grow on one or more petroleum products and to acquire one or more industrially useful characteristics. In one embodiment, the organism is a microorganism, such as further described below.

[0041] An industrially useful characteristic can include any characteristic that enhances biodegradation of petroleum or petroleum products. In one embodiment, an industrially useful characteristic is enhanced biodegradation by an EMO evolved from a microorganism known to biodegrade petroleum or complex hydrocarbon. In another embodiment, an industrially useful characteristic is an ability to grow on two or more complex hydrocarbons. In another embodiment, an industrially useful characteristic is increased rate of cell division in a medium comprising petroleum. In another embodiment, an industrially useful characteristic is an ability to tolerate increased amount of petroleum in a medium. In another embodiment, an industrially useful characteristic is an ability to tolerate increased amounts of petroleum and salt in a medium. In one embodiment, the salt is magnesium. In another embodiment, the salt is calcium. In another embodiment, the salt is potassium. In another embodiment, the salt is sodium. In one embodiment, the concentration of sodium ion is about 0.47 mol/kg. In another embodiment, the concentration of magnesium ion is about 0.053 mol/kg. In one embodiment, the concentration of chloride ion is about 0.55 mol/kg. In one embodiment, the concentration of potassium ion is about 0.01 mol/kg. In one embodiment, the concentration of calcium ion is about 0.01 mol/kg.

[0042] In one embodiment, the growth rate of an evolved organism is enhanced by more than about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 50, 45, 50, 75, or 100 times, as compared to an organism (e.g., wild-type organism, or organism from which the evolved organism was derived from) that had not been evolved. In another embodiment, the evolved organism biodegrades wider varieties of complex hydrocarbons as compared to an organism (e.g., wild-type organism, or organism from which the evolved organism was derived from) that had not been evolved. In one embodiment, the rate of biodegradation of one or more complex hydrocarbon in an evolved organism is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 50, 45, 50, 75, or 100 times higher than that of an non-evolved organism (e.g., wild-type organism, or organism from which the evolved organism was derived).

[0043] An organism can be evolved to gain a characteristic including, but not limited to, enhanced growth in the presence of nitrogen, oxygen, or sulfur sources found in petroleum. In another embodiment, an organism is evolved to gain a characteristic including, but not limited to, enhancing growth in the presence of various metals found in petroleum. In another embodiment, an organism is evolved to gain a characteristic including, but not limited to, enhancing growth in the presence of various salts or elements and found in seawater. Elements found in seawater include, but are not limited to, Hydrogen, Oxygen, Sodium, Chlorine, Magnesium, Sulfur, Potassium, Calcium, Bromine, Molybdenum, Ruthenium, Rhodium, Silver, Palladium, Argentum, Cadmium, Indium, Stannum, Tin, Antimony, Helium, Lithium, Beryllium, Boron, Carbon, Nitrogen, Fluorine, Neon, Aluminium, Silicon, Phosphorus, Argon, Scandium, Titanium, Vanadium, Chromium, Manganese, Ferrum, Iron, Cobalt, Nickel, Tellurium, Iodine, Xenon, Cesium, Barium, Lanthanum, Cerium, Praesodymium, Neodymium, Samarium, Europium, Gadolinium, Terbium, Dysprosium, Holmium, Erbium, Thulium, Ytterbium, Lutetium, Hafnium, Copper, Zinc, Gallium, Germanium, Arsenic, Selenium, Krypton, Rubidium, Strontium, Yttrium, Zirconium, Niobium, Tantalum, Tungsten, Rhenium, Osmium, Iridium, Platinum, Aurum, Gold, Mercury, Thallium, Lead, Bismuth, Thorium, Uranium, and Plutonium.

[0044] In one embodiment, a device for continuous culture is used to culture an organism disclosed herein, such as an evolving organism, EMO, or genetically modified organism. In one embodiment, the organism is a microorganism. In one embodiment, the microorganism is a bacterium, yeast, an alga, or a fungus. The culturing can be continuous ad infinitum, without causing wall growth problem. This technology allows circumventing culture problems known to be associated with wall growth and thus enable efficient and faster evolution of a microorganism toward enhancement or acquisition of industrially desirable traits through evolutionary process.

[0045] In another embodiment, continuous culturing of an organism is carried out without any fluid transfer, including sterilization or rinsing functions. In one embodiment, a continuous culture is achieved inside a flexible sterile tube filled with growth medium. In one embodiment, the medium and the chamber surface are static with respect to each other, and both are regularly and simultaneously replaced by peristaltic movement of the tubing through "gates", or points at which the tube is sterilely subdivided by clamps that prevent the cultured cells from moving between regions of the tube. UV gates can also (optionally) be added upstream and downstream of the culture vessel for additional security.

[0046] In another embodiment, a continuous culture can select continually, rather than periodically, against adherence of dilution-resistant variants to the chemostat surfaces, as replacement of the affected surfaces occurs in tandem with the process of dilution.

[0047] In another embodiment, the flexible sterile tube employed in a continuous culture is subdivided in a transient way such that there are regions that contain saturated (fully grown) culture, regions that contain fresh medium, and a region between these two, wherein one or more chambers referred to as growth or culture chambers are present to form a growth chamber region in which grown culture is mixed with fresh medium to achieve dilution. The gates are periodically released from one point on the tube and replaced at another point, such that grown culture along with its associated growth chamber surface and attached static cells, is removed by isolation from the growth chamber and replaced by both fresh medium and fresh chamber surface.

[0048] In another embodiment, a continuous culture can proceed by repetitive movements of the gated regions of tubing. In one embodiment, this involves simultaneous movements of the gates, the tubing, the medium, and any culture within the tubing. The tubing moves in the same direction; unused tubing containing fresh medium moves into the growth chamber and mix with the culture remaining there, providing the substrate for further growth of the cells contained therein. In one embodiment, this medium and its associated tubing is maintained in a sterile condition by separation from the growth chamber by the upstream gates before introduction into the growth chamber region. Used tubing containing grown culture is simultaneously moved "downstream" and separated from the growth chamber by the downstream gates. As used herein, "upstream" refers to a portion of tubing containing fresh medium and "downstream" refers to a portion of tubing containing used medium.

[0049] In another embodiment, the boundaries between upstream chamber and the growth chamber or between the growth chamber and downstream chamber are defined by gates located along the tube. Gates can be operated as clamps, either opening or closing off a section of tubing. Gates configuration, i.e., where they are located or the number of gates, or the distance between gates can be adjusted according to species-specific demand of a culture. In a given configuration, gates can be designed through one chain of multiple teeth simultaneously moved or in another configuration separated in distinct synchronized. Gates can comprise a system made of two teeth pinching the tubing in a stacking manner.

[0050] In another embodiment, when one or more growth chambers are present, the growth chambers can be used for the same or different purpose. For example, living cells could be grown in a first growth chamber and a second growth chamber with the same or different conditions. In one embodiment, a first growth chamber can be used to grow cells and a second growth chamber can then be used to treat the living cells under different conditions. For example, the cells can be treated to induce the expression of a desired product. Components or additives of the culture medium itself can be added prior to or after the culture begins. For example, all components or additives could be included in the media before beginning the culture, or components can be injected into one or more of the growth chambers after the culture have been initiated.

[0051] In another embodiment, aeration (gas exchange), is achieved directly and without mechanical assistance by the use of gas permeable tubing. In one embodiment flexible gas permeable tubing is made from silicone. Aeration could be achieved through exchange with the ambient atmosphere or through exchange with an artificially defined atmosphere (liquid or gas) that contacts the growth chamber or the entire chemostat. In embodiments where anaerobiosis is desired, the flexible tubing can be gas impermeable. For example, flexible gas impermeable tubing can be made of coated or treated silicone.

[0052] In another embodiment, anaerobic evolution conditions can be achieved by confining regions of the tubing in a specific and controlled atmospheric area to control gas exchange dynamics. This can be achieved either by making the thermostatically controlled box gastight and then injecting neutral gas into it or by placing the complete device in an atmosphere controlled room.

[0053] In another embodiment, the growing chamber can be depressurized or over pressurized according to conditions desired. Different ways of adjusting pressure can be used, for instance applying vacuum or pressurized air to the fresh medium and tubing through its upstream extremity and across the growth chamber; another way of depressurizing or over pressurizing tubing can be done by alternate pinching and locking tubing upstream of or inside the growth chamber.

[0054] In one embodiment, a continuous culture described herein can use tilting movements of the device, and/or shaking of the growth chamber by an external device to decrease aggregation of cells within the growth chamber. Alternatively, one or several stirring bars can be included in the tubing filled with fresh medium before sterilization and magnetically agitated during culture operations.

[0055] In one embodiment, continuous culture described herein can use any form of liquid or semi-solid material as a growth medium.

[0056] In another embodiment, continuous culture described herein can contain multiple growth chambers, such that the downstream gates of one growth chamber become the upstream gates of another. This could, for example, allow one cell to grow alone in the first chamber, and then act as the source of nutrition for a second cell in the second chamber.

[0057] In another embodiment, continuous culture described herein can use an emitter to subject the cells, permanently or temporarily, to one or more of radio waves, light waves, UV-radiation, x-rays, sound waves, an electromagnetic field, a radioactive field, radioactive media, or combinations thereof. The growth chamber region of the device can be subjected to, permanently or temporarily, a different gravitational force. For example, the cells can be grown in a microgravity environment.

[0058] In one embodiment, a device for culturing an organism described herein comprises a) a flexible, sterile tube containing culture medium, b) a system of clamps, each capable of open and closed positions, the clamps being positioned so as to be able to divide the tube into separate regions containing spent culture (downstream region), growing culture (growth chamber), and fresh growth medium (upstream region), c) a component for moving the clamps and the tubing such that a portion of the growth chamber and the associated culture can be clamped off and separated from the growth chamber, and such that a portion of fresh tubing containing unused medium can be joined with a portion of the culture and associated medium already present in the growth chamber, wherein each of the clamps does not move with respect to the tube when said clamp is in the closed position. In one embodiment, the tubing is flexible to allow clamping and segregation into separated chambers. In another embodiment, the tubing is gas permeable, for example comprised primarily of silicon, to allow gas exchange between the cultured organism and the outside environment, according to the conditions desired. In yet another embodiment, the tubing is gas impermeable, to prevent gas exchange between the tubing and the outside environment, if anaerobiosis is desired. In one embodiment, tubing is transparent or translucent, to allow the measurement of turbidity. In yet another embodiment, the growth chamber tubing and associated media and culture can be depressurized or over pressurized relative to ambient atmosphere as desired. In one embodiment, the tubing allows the measure of pH of medium by inclusion of a pH indicator in the tubing composition or lining. In one embodiment, the growth chamber tubing and associated media and culture can be heated or cooled as appropriate. In one embodiment, the growth chamber tubing and associated media and culture can be kept motionless or agitated by any already known method. In one embodiment, the tubing can include one or several stirring bars for agitation purpose. In another embodiment, regions of the tubing can be confined in a specific and controlled atmospheric area to control gas exchange dynamics. In another embodiment, the growth chamber tubing and associated media and culture can be tilted either downward to remove aggregated cells, or upward to remove air.

[0059] In yet another embodiment, a device for culturing an organism described herein comprises a continuous length of flexible, sterile tubing; a system of clamps positioned at points along a section of the tubing, each of the clamps being positioned and arranged so as to be able to controllably pinch the tubing by putting said clamp into a closed position in which the tubing is divided into separate regions on respective sides of said clamp, the separate regions on respective sides of the clamp being merged back into a single region when the clamp is returned to an open position; wherein the clamps and tubing are arranged so that the tubing is clamped at first through fourth points along the tubing, defining first through third regions downstream of the first through third points, respectively; and wherein a volume of the second region delimited by said points two and three is greater than a volume of the first and third regions, wherein the system of clamps is constructed so that, in a repeating pattern, the tubing is clamped upstream of the first point, the tubing is clamped at a point between the second and third points, and the second point is returned to the open position, thereby subdividing the second region into an upstream portion and a downstream portion, merging the first region and the upstream portion, and thereby defining new first through fourth points and first through third regions. In one embodiment, the tubing is gas permeable. In one embodiment, the tubing is gas impermeable. In one embodiment, the tubing is translucent. In one embodiment, the tubing is transparent. In one embodiment, the contents of the tubing in the second region can be controllably depressurized or over pressurized relative to ambient atmosphere. In one embodiment, the device further comprises a pH indicator in the tubing, a heating and cooling device that can control a temperature of contents of the tubing, an agitator, or a combination thereof. In one embodiment, the agitator comprises at least one stirring bar. In one embodiment, the regions of the tubing can be confined in a specific and controlled atmospheric area to control gas exchange dynamics. In another embodiment, the device can further comprise a device to control tilting of the second portion of the tubing.

[0060] In yet another embodiment, a device for culturing an organism described herein comprises flexible tubing containing culture medium; and a system of clamps, each capable of open and closed positions, the clamps being positioned so as to be able to divide the tubing into: i) an upstream region containing unused culture medium; i) a downstream region containing spent culture medium; and iii) a growth chamber region for growing said cells disposed between the upstream and downstream regions; wherein the system of clamps is constructed and arranged to open and close so as to clamp off and define the growth chamber region of the tubing between the upstream and downstream regions of the tubing, and to cyclically redefine the growth chamber region of the tubing so that a first portion of the previously defined growth chamber region becomes a portion of the downstream region of the tubing, and a portion of the previously defined upstream region of the tubing becomes a portion of the growth chamber region of the tubing. In one embodiment, the system of clamps is structured and arranged so that each of the clamps does not move with respect to the tubing when said clamp is in the closed position. In one embodiment, the tubing is gas permeable. In one embodiment, the tubing is gas impermeable. In one embodiment, tubing is one of transparent and translucent to permit a turbidity meter to determine the density of the culture. In yet another embodiment, the device further comprises a pressure regulator constructed to change a pressure of the growth chamber portion of the tubing relative to ambient pressure. In one embodiment, the tubing comprises a pH indicator. In yet another embodiment, the device further comprises a temperature regulator constructed to control the temperature of the growth chamber region of the tubing, an agitator constructed to allow agitation of the growth chamber portion of the tubing, or a combination thereof. In one embodiment, the agitator comprises at least one stirring bar. In yet another embodiment, the growth chamber region comprises one or more growth chambers containing culture medium.

[0061] In one embodiment a microorganism is evolutionary modified to produce higher amounts of an end-product (e.g., fatty acids or ethanol) than the same strain of non-evolutionary modified

microorganism by culturing it with one or more specific carbonaceous materials.

Genetic Engineering

[0062] A microorganism suitable for methods described herein can be naturally occurring or genetically modified. A genetically engineering an organism refers to directly manipulating one or more genes of an organism, such as through molecular cloning and recombinant DNA technology. Genetic engineering or other methods have been utilized to create or improve these oil-degrading microorganisms (e.g., U.S. Pat. Nos. 6, 1 10,372, or 4,535,061, each of which is herein incorporated by reference in its entirety). In another embodiment, an organism is evolutionarily modified and is subsequently genetically modified through genetic engineering. In one embodiment, an EMO is selected and genetically engineered to further acquire one or more industrially useful characteristics. In one embodiment an EMO that is adapted to grow on a particular complex hydrocarbon is genetically engineered to increase its growth rate. In another embodiment, an EMO that is adapted to biodegrade petroleum or petroleum products is genetically engineered to increase its rate of biodegradation.

[0063] In another embodiment, an organism can be genetically engineered and then subsequently evolutionarily modified, such as for better growth rate, a higher rate of biodegradation, or both. In one embodiment, an organism can be genetically engineered to metabolize one or more complex hydrocarbons (such as petroleum) and evolved to increase its tolerance to seawater.

[0064] In yet another embodiment, an organism can be genetically engineered to have one or more characteristics of an EMO. For example, an EMO can be selected and its genome, or a portion thereof is analyzed, such as by sequencing or other techniques, to identify one or more genetic modifications occurred in the genome of an EMO. The identified genetic modifications can then be expressed in another organism through genetic engineering, such as by recombinant DNA techniques. For example, an EMO can have a mutation in a gene. The mutation is identified and cloned into another organism or a wild-type or non-EMO. The mutation can be inserted into an expression cassette designed for the chosen organism and introduced into the host where it is recombinantly produced. The choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer, is within the level of skill of the one ordinarily skilled in the art. The resultant molecule, containing the individual elements linked in proper orientation and reading frame, may be inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms, such as for bacteria (see, e.g., Studier and Moffatt. 1986. J. Mol. Biol. 189: 1 13; Brosius. 1989. DNA 8: 759, for E. coli), algae (see e.g., Mayfield, S. P., S. E.

Franklin, and R. A. Lerner. 2003. Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci USA 100:438-42, Zaslavskaia, L. A., J. C. Lippmeier, C. Shih, D. Ehrhardt, A. R. Grossman, and K. E. Apt., 2001. Trophic conversion of an obligate photoautotrophic organism through metabolic engineering. Science 292:2073-5) and yeast (see, e.g., Schneider and Guarente. 1991. Meth. Enzymol. 194: 373).

Organism for Evolutionary Modification

[0065] In one embodiment, an organism that is evolutionarily modified is a unicellular organism. In another embodiment, an organism that is evolutionarily modified is a multicellular organism. In another embodiment, the organism is a prokaryote. In yet another embodiment, the organism is a eukaryote. In one embodiment, the organism is a microorganism. In one embodiment, the microorganism is adapted to grow on carbon and/or nitrogen sources available in petroleum or petroleum products. A suitable microorganism can be a eukaryote or prokaryote, being either a bacterium, a fungus, a yeast, an alga, plant cell or an animal cell. In one embodiment, the microorganism is a bacterium. In another embodiment, the microorganism is a yeast. In another embodiment, the microorganism is a fungus. In another embodiment, the microorganism is an alga.

[0066] In another embodiment a microorganism utilized herein is evolutionarily modified to serve as an improved bioagent for biodegrading complex hydrocarbons.

[0067] In another embodiment a microorganism can be a strain different than its wild type, which can be identified by the mutations acquired during the course of culture, and these mutations, can allow the new cells to be distinguished from their ancestor's genotype characteristics. Thus, one can select new strains of microorganisms by segregating individual strains with improved rates of reproduction through the process of natural selection.

[0068] In another embodiment a microorganism can be evolutionarily modified in a number of ways so that their growth rate, viability, and utility as an environmentally friendly biocontainment agent can be improved. For example, a microorganism can be evolutionarily modified to enhance its ability to grow on a particular substrate, to use a particular complex hydrocarbon, to use a particular inorganic source, to use a particular sulfur source, to use a particular energy source, to use a particular nitrogen source, to tolerate a component of a substrate/material/source that could be inhibiting growth, or to tolerate specific environmental conditions (such as high or low pH, salinity, pressure, temperature or oxygenation).

[0069] While a variety of species can be used for evolutionary modification, a marine bacterium is useful for adapting to increased rate of oil- degradation. In one embodiment, a marine bacterium is a bacterium known for its oil-degrading capability. In another embodiment, a marine bacterium is a bacterium not known to degrade oil in marine environment. In another embodiment, a bacterium is selected from Acinetobacter johnsonii, Acinetobacter haemolyticus, Alcanivorax borkumensis, Alcanivorax dieselolei, Alcanivorax jadensis, Alcanivorax venustensis, Azoarcus toluvorans, Desulfatibacillum aliphaticivorans, Desulfotomaculum sp., Gordonia alkanivorans, Marinobacter hydrocarbonoclasticus, Neptunomonas naphthovorans, Novosphingobium aromaticivorans, Rhodococcus erythropolis, Rhodococcus

baikoneurensis, Sphingobium yanoikuyae, Sphingomonas sp., Tranquillimonas alkanivorans,

Tropicimonas isoalkanivorans, Thauera aromatica, and Thalassolituus oleovorans.

[0070] Other microorganisms that can be useful for evolving to acquire biodegradation characteristics include species of the following genera in the Pseudomonadaceae family comprising gram-negative aerobic rods and cocci: Pseudomonas; Variovorax; Chryseobacterium; Comamonas; Acidovorax;

Stenotrophomonas; Sphingobacterium; Xanthomonas; Frateuria; Zoogloea; Alcaligenes;

Flavobacterium; Derxia; Lampropedia; Brucella; Xanthobacter; Thermus; Thermomicrobium;

Halomonas; Alteromonas; Serpens; Janthinobacterium; Bordetella; Paracoccus; Beijerinckia; and Francisella; species of the following genera in the Nocardioform Actinomycetes family comprising gram- positive Eubacteria and Actinomycetes: Nocardia; Rhodococcus; Gordona; Nocardioides;

Saccharopolyspora; Micropolyspora; Promicromonospora; Intrasporangium; Pseudonocardia; and Oerskovia; species of the following genera in the Micrococcaceae family comprising gram-positive cocci: Micrococcus; Stomatococcus; Planococcus; Staphylococcus; Aerococcus; Peptococcus;

Peptostreptococcus; Coprococcus; Gemella; Pediococcus; Leuconostoc; Ruminococcus; Sarcina; and Streptococcus; species of the following genera in the Vibrionaceae family comprising facultative anaerobic gram-negative rods: Aeromonas; Photobacterium; Vibrio; Plesiomonas; Zymomonas;

Chromobacterium; Cardiobacterium; Calymmatobacterium; Streptobacillus; Eikenella; and Gardnerella; species of the following genera in the Rhizobiaceae family comprising gram-negative aerobic rods and cocci: Phyllobacterium; Rhizobium; Bradyrhizobium; and Agrobacterium; species of the following genera in the Cytophagaceae family comprising non-photosynthetic, gliding bacteria, non- fruiting: Cytophaga; Flexibacter; Saprospira; Flexithrix; Herpetosiphon; Capnocytophaga; and Sporocytophaga; or species of the following genera in the Corynebacterium family comprising irregular, non-sporing gram-positive rods: Aureobacterium; Agromyces; Arachnia; Rothia; Acetobacterium; Actinomyces; Arthrobactera; Arcanobacterium; Lachnospira; Propionibacterium; Eubacterium; Butyrivibria; Brevibacterium;

Bifidobacterium; Microbacterium; Caseobacter; and Thernoanaerobacter .

[0071] In one embodiment, a microorganism is evolved to robustly grow in various pH. Non-limiting examples of pH include about 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, or 9.6.

[0072] In one embodiment, a microorganism is evolved to robustly grow in various temperatures. Non- limiting examples of temperature include about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 1 1°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C. [0073] In one embodiment, a microorganism is evolved to robustly grow in various pressures. Non- limiting examples of pressure include about 760 mmHg, 774 mmHg, 788 mmHg, 802 mmHg, 816 mmHg, 830 mmHg, 845 mmHg, 859 mmHg, 874 mmHg, 890 mmHg, and 904 mmHg.

[0074] In one embodiment, an evolved microorganism is identified and selected for its robust growth in a medium comprising petroleum. In one embodiment, robust growth is confirmed by visually inspecting the turbidity of the culture. Non-limiting examples of turbidity include optical density of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.

[0075] In another aspect an EMO is used to breakdown or disperse a complex hydrocarbon mixed with water. In one embodiment the complex hydrocarbon is petroleum. In one embodiment the EMO uses the complex hydrocarbon as an energy source. In another embodiment the EMO is used to breakdown or disperse a complex hydrocarbon mixed with fresh water. In another embodiment the EMO is used to breakdown or disperse a complex hydrocarbon mixed with sea water. In another embodiment the EMO is used to breakdown or disperse a complex hydrocarbon treated with a dispersant. In another embodiment the EMO is used to breakdown or disperse a complex hydrocarbon as part of a bioremediation program. In another embodiment the EMO is used to breakdown or disperse a complex hydrocarbon such as petroleum that has contaminated a beach, wetlands, river bottom, mudflats, stream, river, sea, or ocean. In another embodiment the EMO is used to breakdown or disperse a complex hydrocarbon such as petroleum present in a coastal region, pelagic region, surface region, or as a deep water oil plume.

[0076] In one embodiment an EMO is able to breakdown or disperse a complex hydrocarbon such as petroleum faster and/or more efficiently than indigenous microflora. In another embodiment the density or numbers of an EMO decline after they have broken down complex hydrocarbons that are present. In another embodiment any remaining EMOs are incorporated into the surrounding ecosystem.

Evolutionarily Modified Community of microbial strains (EMCMS)

[0077] In one embodiment, one or microbes are selected for their known properties. In one embodiment, a known property is a biological property, such as preference toward certain nutrients. In another embodiment, a known property is a commercially useful property, such as digesting a chemical used in industry. Examples of a known property include, but are not limited to, tropism toward food source or growth condition, tolerance toward overcrowding or high-density growth, tolerance toward certain environmental condition, genomic stability of instability in the presence of a mutagen, tolerance toward UV, radiation or other mutagens, rate of cell division, or other properties affecting the survival of a microbe in a non- indigenous environment.

[0078] In one embodiment, one or more selected microbes are pooled together as a candidate for a community of microbial strains (CMS). In one embodiment, the CMS is formed by culturing selected microbes together as a pool and by removing incompatible strains from the community. Exemplary sources of incompatibility includes, but is not limited to, dominating the community by outgrowing other strains in the community, killing or inhibiting other strains in the community, producing pathogens or molecules inhibiting the growth of other strains, frequently fusing with other strains, causing increased mutations in other strains, depriving food sources other strains are critically depending on, or reducing the diversity of the community.

[0079] In one embodiment, the CMS comprises microorganisms derived from a single environment. Exemplary environments include, without limitation, sea, lake, river, landfill, mud, sand, groundwater, or wetland. In one embodiment, the CMS comprises microorganisms not derived from a single environment. In further embodiments, the CMS comprises genetically modified microbial strains. In another further embodiment, the CMS comprises evolutionarily modified microbial strains.

[0080] In one embodiment, the CMS is cultured under the optimal growth condition of one of the community strains. In another embodiment, the CMS is cultured under a condition foreign to any of the community strains. In one embodiment, a foreign condition is a non-indigenous condition of any of the community strain. In another embodiment, a foreign condition is an artificial condition that does not exist in nature. In another embodiment, a foreign condition is a condition mimicking an environmental condition. In another embodiment, a foreign condition is a condition mimicking one or more aspects of a natural environmental condition. In another embodiment, a foreign condition is a condition mimicking one or more aspects of an industrial environmental condition.

[0081] In one embodiment, an industrial environmental condition is oil contamination. In one embodiment, an oil-contaminated condition is contamination of water. In another embodiment, an oil- contaminated condition is contamination of seawater. In another embodiment, an oil-contaminated condition is contamination of underground water. In another embodiment, an oil-contaminated condition is contamination of soil.

[0082] In one embodiment, an industrial environmental condition is a condition comprising marine pollutant. Examples of marine pollutant includes, but is not limited to, PCBs, DDT, pesticides, furans, dioxins, phenols, radioactive waste, or heavy metals such as mercury, lead, nickel, arsenic or cadmium.

[0083] In one embodiment, the CMS is cultured for an extended period time to evolutionarily adapt to a foreign condition. In one embodiment, an evolutionarily modified CMS (EMCMS) is a CMS cultured for about 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or 53 weeks in a continuous culture system described herein. In another embodiment, an EMCMS is a CMS cultured for about 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, 10.5 years, 1 1 years, 1 1.5 years, 12 years, or 12.5 years in a continuous culture system described herein. In one embodiment, a CMS is cultured continuously without interruption in the system described herein for the periods described herein. In another embodiment, a CMS is cultured with intermittent interruption for the periods described herein. [0084] In one embodiment, a CMS is cultured while various aspects of a defined foreign condition is gradually introduced. In one embodiment, a CMS is cultured for a period under the temperature defined for a particular foreign condition. In one embodiment, a CMS cultured for a period under a defined foreign temperature is cultured under a pH defined for a particular foreign condition. In another embodiment, other conditions representing a defined foreign condition, such as types and amounts of nutrients, are gradually introduced to a CMS culture that has been adapted to grow at a particular temperature or pH or both.

[0085] In another embodiment, a CMS is introduced directly into a foreign condition and the surviving members of the CMS are taken as EMCMS emerged from the CMS. In another embodiment, EMCMS is continuously cultured in the same foreign condition and sampled intermittently. In one embodiment, the samples are tested for the community's adaptability to the foreign condition. Adaptability is measured by, for example, optical density, growth rate, pulse-chasing using labeled nutrients, measuring expression level of metabolic enzymes, or genotyping.

[0086] In one embodiment, an EMCMS is introduced to seawater contaminated with oil. In one embodiment, the EMCMS is introduced in a small amount distributed to various entry points strategically positioned to cover a vast area of seawater. In another embodiment, the EMCMS is introduced in to a single point of the contaminated area. In another embodiment, the EMCMS is scaled up in a bioreactor and continuously pumped into the contaminated area. In another embodiment, the EMCMS is scaled up to an amount sufficient enough to control the contamination in the entire area within a short period of time. In one embodiment, the short period of time is about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 40 days. In another embodiment, the EMCMS is sprayed over the contaminated area.

[0087] In one embodiment, an EMCMS comprises two or more strains selected from the group consisting of Alcanivorax borkumensis, Alcanivorax dieselolei, Alcanivorax jadensis, Azoarcus toluvorans, Desulfatibacillum aliphaticivorans, Desulfotomaculum sp., Gordonia alkanivorans,

Marinobacter hydrocarbonoclasticus, Neptunomonas naphthovorans, Novosphingobium aromaticivorans, Rhodococcus erythropolis, Sphingobium yanoikuyae, Sphingomonas sp., Tranquillimonas alkanivorans, Tropicimonas isoalkanivorans, Thauera aromatica, and Thalassolituus oleovorans. In another embodiment, an EMCMS comprises Alcanivorax borkumensis, Alcanivorax dieselolei, Alcanivorax jadensis, Azoarcus toluvorans, Desulfatibacillum aliphaticivorans, Desulfotomaculum sp., Gordonia alkanivorans, Marinobacter hydrocarbonoclasticus, Neptunomonas naphthovorans, Novosphingobium aromaticivorans, Rhodococcus erythropolis, Sphingobium yanoikuyae, Sphingomonas sp.,

Tranquillimonas alkanivorans, Tropicimonas isoalkanivorans, Thauera aromatica, and Thalassolituus oleovorans. In another embodiment, an EMCMS comprises Alcanivorax borkumensis, Desulfatibacillum aliphaticivorans, Gordonia alkanivorans, Marinobacter hydrocarbonoclasticus, Neptunomonas naphthovorans, Novosphingobium aromaticivorans, Rhodococcus erythropolis, Sphingobium yanoikuyae, Tranquillimonas alkanivorans, Tropicimonas isoalkanivorans, Thauera aromatica, and Thalassolituus oleovorans. [0088] In one embodiment, an EMCMS is streaked on a agar plate and a colony from each strain is obtained. In one embodiment, cells from each obtained colony are pooled and genotyped. In another embodiment, cells from each obtained colony are pooled and genomic DNAs are extracted for sequencing the genome. In another embodiment, genes modified in EMCMS are identified based on the sequence information obtained from the genomes of EMCMS. In another emobodiment, expression profile of EMCMS is determined by measuring the quantities and types of mRNAs expressed in EMCMS. In another embodiment, proteins uniquely or robustly expressed in EMCMS are identified in a high- throughput protein screening methods known in the art (e.g., 2-dimnesional gel electrophoresis, antibody array, or mass spectrometry).

[0089] In one embodiment, two or more EMCMS are combined together and cultured continuously. In one embodiment, combined EMCMS are tested for synergistic bioremediational effects on contaminated area. In another embodiment, combined EMCMS are tested for bioremediational effects unseen with EMCMS prior to the combination. A barriage of carbon sources are utilized in an in vitro test to identify utilization of any new carbon source or increased rate of utilization thereof. In one embodiment, the test is performed in a high-throughput setting.

EXAMPLES

Example 1

[0090] A sample is obtained from Gulf seawater contaminated with dispersed oil was taken from a pelagic area. A microorganism was cultured in the following media: Gulf of Mexico Sea Water (28.991 N, 89.142 W) contaminated with Deepwater Horizion Crude Oil + Dispersant, 20mg/L (NH4)2S04, lOmg/L K2HP04, lOmg/L FeS04 ^« 7H20. The culture was placed in a continuous culture apparatus as described in U.S. Patent Application Publication No. 20070037276 and U.S. Patent No. 7,939,315.

Overnight cultures of oil-degrading microorganisms were spun down and are resuspended in 3mL non- autoclaved seawater from the same bucket the oil that was taken from and injected into the growth chamber of the apparatus. The culture was maintained at 28°C. Air was provided as gas bubble. Other parameters include the following: 14 second balancing and 30 second stop time to agitate the solution. Example 2

[0091] An oil sample containing oil and Corexit™ dispersant is obtained. One part of the sample is mixed with 9 part of a media. A microorganism is added to the sample-media mixture. The

microorganism is culture at 28°C continuously. A small portion of culture is taken out and used as an inoculum to a sample-media mixture having the ratio of 2:8, 3 :7, 4:6. 5 :5, 6:4, 7:3, 8:2, and 9: 1. A culture robustly growing in sample-media mixed with a ratio of 9: 1 is taken and is inoculated to a medium comprising only oil and Corexit™.

Example 3

[0092] An EMO, is evolved to breakdown crude oil is applied to a crude oil spill comprising crude oil, seawater and a Corexit™ dispersant. The EMO uses the crude oil as its primary energy source and digests the crude oil.

Example 4 [0093] A community of microbial strain was selected and tested for rapid biodegradation of hydrocarbon contaminants originated from crude oil spill in seawater, Actual samples of oil-contaminated seawater from the Gulf of Mexico was obtained. Samples were collected near the mouth of the South Pass of the Mississippi River delta. The samples contained oil that has been partially solubilized with dispersant. The samples were fed into a continuous culture system described in U.S. Patent Application Publication No. 20070037276 and U.S. Patent No. 7,939,315: culture tubing was filled with the seawater/oil/dispersant mixture, sterilized and loaded onto the culture system. No other carbon or energy source was introduced in the culture chamber other than the oil/dispersant.

[0094] To mimic the environmental conditions where the oil was found, the culture system was set to mimic the environmental condition of sea surface on which the oil was collected. A large bubble of air was included in the culture chamber to mimic ambient oxygen concentrations at the surface. The temperature was set to 28°C, which was approximately the average temperature of surface seawater in the Gulf during summer. Mild agitation of the culture chamber was introduced to mimic the action of waves.

[0095] To select microbes for adaptive growth on oil, the culture chamber was initially inoculated with non-sterilized Gulf seawater. This seawater contains any number of indigenous microbes (oil-eating and otherwise) as well as any virus and predators capable of affecting the composition of the eventual community. It was then inoculated with live cultures of a variety of oil-degrading microbes chosen for their ability to degrade different components of crude oil. Emphasis was placed on microbes known to be able to survive in marine environment. A complete list of microbial strains chosen for the study is shown in Table 1. DSMZ Strain Number refers to strain identification number of DSMZ, GmbH (German collection of microorganisms and cell culture).

Table 1. Microbial strains.

Novosphingobium aromaticivorans 12444

Thauera aromatica 6984

Thalassolituus oleivorans 14913

Azoarcus toluvorans 1 5124

[0096] After inoculation of strains into the growth chamber, an initial decline in optical density over the course of the first few days was observed. This is indicative of a rapid die-off of microbes due to an inability to survive under the conditions inside the growth chamber or on the oil/dispersant mixture as the sole source of carbon and energy. After one week, growth in the culture chamber was observed. At three weeks, growth significant enough to take a sample of the community was observed. The 3-week sample was tested for its ability to grow on a seawater/oil sample taken from the Gulf at 28°C in flasks. For the test, three samples were prepared. Each sample was tested in triplicate. Sample No. 1 contained only oil/seawater and was inoculated with a sample of non-sterile seawater. This sample was designed to mimic natural attenuation (or doing nothing). Sample No. 2 was identical to sample No. 1, except that 20 mg/L ammonium sulfate, 10 mg/L potassium phosphate and 10 mg/L iron chloride were added as nutritional supplements. This sample was designed to mimic biostimulation. Sample No. 3 was identical to sample No. 2 except that it was also inoculated with a sample of community of microbial strain taken from the continuous culture system at week 3. The same experiment was performed using the community of microbial strains taken from the culture system at week 5 to monitor progress. FIG. 2A shows the growth curves (average and standard deviation) for each sample at 5 weeks. Growth in sample No. 3, but not in samples No. 1 and No. 2 was observed, indicating that community of microbial strains adapted for 3 weeks in the continuous culture system is capable of growing (and, consequently degrading) oil in Gulf seawater. Error bars indicate ± 1 std deviation. FIG. 2B shows the turbidity in the flask for sample No. 3 and the clarity of the flasks for samples No. 1 and No. Photos in FIG. 2B are representative flasks from the experiment. FIG. 3 shows the growth curves for the community of microbial strains taken after 3 weeks and after 5 weeks. The result suggests that the community of microbial strains that has been adapting to the seawater/oil/dispersant mixture at 28°C for a longer period of time grows faster and to a higher cell density. This indicates progressively better adaptation is occurring over time. Average cell densities are plotted versus time. Error bars indicated ±1 standard deviation.

[0097] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.