Microalgae useful in methods for reducing methane production

Abstract

The present disclosure features methods for modulating the production of a small halogenated organic compound from a microalgae, as well as related compositions and methods thereof.

Description

[0001] MTCROALGAE USEFUL IN METHODS FOR REDUCING METHANE PRODUCTION CLAIM OF PRIORITY

[0002] This application claims priority to U. S. Application No. 63 / 712,031, filed October 25, 2024; the entire contents of the foregoing application is incorporated herein by reference.

[0003] BACKGROUND

[0004] There are growing concerns about the greenhouse gas emissions from animal agriculture. A large portion of these emissions can be attributed to biogenic enteric methane (CFL) emissions from all domesticated ruminants (3.2 % of total U. S. emissions; EP A, 2019). Small halogenated organic compounds, such as chloroform, bromochloromethane, and 2-bromoethane sulfonate, may act as inhibitors of enteric methane production (Hristov, A. N. et al. J Anim Sci (2013) 91(11):5045-69). These small organic halogenated compounds can competitively inhibit the activity of methyl -coenzyme M reductase (mMCR), the enzyme that catalyzes the final step of CH4 synthesis by methanogens found in the rumen (Wood, J. M. et al. Biochemistry (1968) 7(5): 1707-1713; Ferry, J. G. Annu Rev Microbiol (2010) 64:3117-3126). However, animal and human safety and environmental concerns prohibit using these small halogenated organic compounds from being applied directly as livestock feed additives. There is therefore a need for developing scalable alternatives for reducing greenhouse gas emissions, e.g., methane, by employing microalgae.

[0005] SUMMARY OF THE INVENTION

[0006] The present disclosure features methods for producing and increasing the production of a small, halogenated organic compound. In some embodiments, the methods described herein comprise preparing a culture of microalgae and acquiring information about the small, halogenated organic compound. In some embodiments, the present disclosure features methods of modulating production of a small halogenated organic compound from a microalgae. In some embodiments, the methods feature modulating production of a small halogenated organic compound from a consortium of microalgae. In some embodiments, the methods described herein comprise preparing a culture of microalgae, contacting the culture with a halogen source and a small organic compound to form a mixture, mixing the mixture, and / or acquiring information about the small halogenated organic compound.

[0007] In an aspect, the methods described herein comprise (i) preparing a culture of microalgae, thereby initiating a halogenation reaction; and ii) acquiring information about the small, halogenated organic compound. In some embodiments the halogenation reaction is an ecological reaction. In some embodiments, the halogenation reaction encompasses biological generation of a small, halogenated organic compound. In some embodiments, the microalgae endogenously produce a small organic compound and a halogen source. In an embodiment, the culture is a monoculture. In an embodiment, the culture is a co-culture, e.g., the co-culture comprises a plurality of microalgae strains.

[0008] The microalgae may be a cyanobacterium, a diatom, a haptophyte, a cryptophyte or a dinoflagellate. In an embodiment, the microalga is selected from Nitzschia cf. pelhicida, Isochrysis galbana, Nitzschia spp., (e.g., Nitzschia sp. 2526), Porosira glacialis, Bigelowellia longifila, Rhodomonas salina, Heterocapsa rotundata, Ditylum brightwellii or a combination thereof. In an embodiment, the microalga is capable of forming a biofdm. In an embodiment, the microalgae comprises a high lipid content, e.g., a lipid content of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In an embodiment, the microalgae comprising a high lipid content is selected from Botryococcus brannii, Isochrysis galbana, Neochloris oleoabimdans, Phaeodactylum tricornutum, Pleurochrysis carterae, Prymnesium parvum, Tetradesmus dimorphns, Tetraselmis chin, Tetraselmis snecica, and Tisochrysis hitea, or a combination thereof.

[0009] The microalgae may be allelopathic or may be capable of producing an allelochemical. In an embodiment, the allelochemical is a small halogenated organic compound. In an aspect, the small halogenated organic compound has a structure of Formula (Z):

[0010] or a salt, tautomer, or isomer

[0011]

[0012] R5a, R5b, and R5cis independently hydrogen, halogen, C1-C6alkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, or heterocyclyl, wherein each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl is optionally substituted with one or more R6, and at least one of Rla, Rlb, Rlc, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, and R5cis independently halogen; R6is halogen, Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -ORA, or -NRBRC; RAis hydrogen, C1-C6alkyl, C1-C6heteroalkyl, or C2-C6alkenyl; RBand RCare each independently hydrogen, C1-C6alkyl, or C1-C6heteroalkyl; each of m and n is independently selected from 0, 1, 2, or 3; and “ - ” is a single or double bond, wherein when - is a double bond, each of R2band R3bis independently absent.

[0013] In an embodiment of Formula (Z), each of Rla, Rlb, and Rlcis independently halogen or hydrogen, wherein at least one of Rla, Rlb, and Rlcis halogen. In an embodiment, the halogen is selected from chlorine, bromine, or iodine. In an embodiment of Formula (Z), each of Rla, Rlb, Rlcis independently halogen or hydrogen, wherein at least two of Rla, Rlb, and Rlcis halogen. In an embodiment, the halogen is selected from two of chlorine, bromine, or iodine. In an embodiment of Formula (Z), each of Rla, Rlb, Rlcis independently halogen. In an embodiment, the halogen is selected from chlorine, bromine, or iodine. In an embodiment of Formula (Z), each of R5a, R5b, and R5cis independently halogen or hydrogen, wherein at least one of R5a, R5b, and R5Cis halogen. In an embodiment of Formula (Z), each of R5a, R5b, and R5cis independently halogen or hydrogen, wherein at least two of R5a, R5b, and R5cis halogen. In an embodiment of Formula (Z), each of R5a, R5b, and R5cis independently halogen. In an embodiment of Formula (Z), - is a single bond. In an embodiment of Formula (Z), each of m and n is independently selected from 0, 1, 2, or 3, and - is a single bond.

[0014] In an embodiment, the small halogenated organic compound is chlorinated; brominated; iodinated; chlorinated and iodinated; chlorinated and brominated; brominated and iodinated; or chlorinated, brominated, or iodinated. In an embodiment, the small halogenated organic compound is brominated. In an embodiment, the small halogenated organic compound comprises 1, 2, or 3 halogen atoms. In an embodiment, the small halogenated organic compound comprises 1, 2, or 3 bromine atoms. In an embodiment, the small halogenated organic compound comprises an acetone moiety. In an embodiment, the small halogenated organic compound comprises dibromomethane, dibromoacetone, bromopentanedione, bromoform, or tribromoacetone. In an embodiment, wherein the small halogenated organic compound comprises dichloroiodomethane, dibromochloromethane, 1,1 -dibromoacetone, bromoacetone, dibromomethane, 3-bromo-2,4-pentanedione, bromoform, 1,3 -tribromoacetone, or 1,1,1 -tribromoacetone. In an aspect, the method further comprises sequestering the small, halogenated organic compound with a microalgae strain. In an embodiment, the sequestering comprises culturing a microalgae strain with a high lipid content, e.g., a lipid content of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., 10-40% lipid content). In an embodiment, the microalgae strain with a high lipid content is Isochrysis galbana.

[0015] In an embodiment of the method for producing a small halogenated organic compound, the acquiring comprises: (i) characterizing the small halogenated organic compound, e.g., by gas chromatography or mass spectrometry; (ii) characterizing the microalgae, e.g., the cell concentration, e.g., by flow cytometry, or cell viability, e.g., by LIVE / DEAD assay; and / or (iii) characterizing the haloperoxidase activity, e.g., by aminophenyl fluorescein (APF) assay.

[0016] The method may further comprise (iv) separating the small halogenated organic compound from the mixture. In an embodiment, the (iv) separating comprises centrifuging the mixture, e.g., centrifuging the mixture into cell pellet and supernatant fractions. In an embodiment, the (iv)separating comprises extracting the small, halogenated organic compound, e.g., by solvent extraction. In an embodiment, the (iv) separating comprises extracting with an oil, e.g., canola oil. In an embodiment, the (iv) separating comprises extracting with a microalga (e.g., a high-lipid content microalgae).

[0017] The cell density of the culture may be between about 102to 106cells / mL, e.g., about 102, 103, 104, 105, or 106cell / mL. In an embodiment, the temperature of the culture is between about 10°C to 50°C, e.g., 20°C, 25°C or 37°C. In an embodiment, the pH of the culture is between about 4 to 10, e.g., about 4, 5, 6, 7, 8, 9, or 10. In an embodiment, the ionic strength of the microalgal culture is between about 0.1 mM to 4 M.

[0018] In an aspect, the present disclosure features methods for reducing production of methane in a rumen community, the method comprising: (i) preparing a culture of microalgae; (iv) separating the small halogenated organic compound from the mixture; and (v) providing the small halogenated organic compound to a rumen community under conditions sufficient to reduce the production of methane. In an embodiment, the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In an embodiment, the methane production is reduced by between 10-75%. In a further embodiment, the present disclosure describes methods for reducing production of methane in a manure management system, the method comprising: (i) preparing a culture of microalgae; (iv) separating the small halogenated organic compound from the mixture; (v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane. In a further embodiment, the methods comprises (i) preparing a culture of microalgae; (ii) contacting the culture with a halogen source and a small organic compound to form a mixture; (iii) mixing the mixture; iv) separating the small halogenated organic compound from the mixture; (v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane. In an embodiment, the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In an embodiment, wherein the methane production is reduced by between 10-75%.

[0019] The present disclosure further recites methods for preparing animal feed for reducing the production of methane in a rumen community, the method comprising: (i) preparing a culture of microalgae; (iv) separating the small halogenated organic compound from the mixture; and (v) preparing a composition comprising a small halogenated organic compound suitable for use as an animal feed, thereby preparing animal feed for reducing the production of methane in a rumen community.

[0020] In another aspect, the present disclosure features methods for reducing production of methane in a rumen community, the method comprising: (i) preparing a culture of microalgae; (ii) contacting the culture with a halogen source and a small organic compound to form a mixture; (iii) mixing the mixture; (iv) separating the small halogenated organic compound from the mixture; and (v) providing the small halogenated organic compound to a rumen community under conditions sufficient to reduce the production of methane. In a further embodiment, the method comprises: (i) preparing a culture of microalgae; and (v) providing the small halogenated organic compound to a rumen community under conditions sufficient to reduce the production of methane. In an embodiment, the method further comprises acquiring a value for the level of methane prior to the contacting of step (ii). In an embodiment, the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In an embodiment, the methane production is reduced by between 10-75%.

[0021] In a further embodiment, the present disclosure describes methods for reducing production of methane in a manure management system, the method comprising: (i) preparing a culture of microalgae; (ii) contacting the culture with a halogen source and a small organic compound to form a mixture; (iii) mixing the mixture;; (iv) separating the small halogenated organic compound from the mixture; (v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane. In an embodiment, the method further comprises acquiring a value for the level of methane prior to the contacting of step (ii). In an embodiment, the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In an embodiment, wherein the methane production is reduced by between 10-75%.

[0022] In a further embodiment, the method comprises reducing production of methane in a manure management system, the method comprising: (i) preparing a culture of microalgae; (iv) separating the small halogenated organic compound from the mixture; (v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane.

[0023] The present disclosure further recites methods for preparing animal feed for reducing the production of methane in a rumen community, the method comprising: (i) preparing a culture of microalgae; (ii) contacting the culture with a halogen source and a small organic compound to form a mixture; (iii) mixing the mixture; (iv) separating the small halogenated organic compound from the mixture; and (v) preparing a composition comprising a small halogenated organic compound suitable for use as an animal feed, thereby preparing animal feed for reducing the production of methane in a rumen community.

[0024] Additional embodiments of the present disclosure are described in further detail herein in the Drawings, Description, Examples, and Claims.

[0025] BRIEF DESCRIPTION OF DRAWINGS FIGS 1A-D. illustrate the haloperoxidase activity, bromoform production, growth, and physical morphology of various microalgae strains. FIG. 1A is a bar graph showing the haloperoxidase activity of several microalgae strains relative to a cell-free medium. FTG. IB is a table showing haloperoxidase activity, relative growth rate, and bromoform production for Porosira glacialis, Nitzschia cf. pellucida, Porphyridium cruentum. Phaeodactylum tricornutiim, and Isochrysis galbana.

[0026] FIG. 2 shows typical flow cytometry results for a co-culture of N. pellucida / I. galbana.

[0027] FIGS. 3A-C demonstrate that microalgae growth of competitor strains is attenuated in the presence of the allelopathic Nitzschia species. FIG. 3A is a graph showing the cell concentration of N. pellucida over the course of 9 days as a monoculture and as a co-culture with Nitzschia sp. 2526 and as a co-culture with I. galbana. FIG 3B is a graph showing the cell concentration of Nitzschia sp. 2526 over the course of 9 days as a monoculture and as a coculture with Nitzschia 0303. FIG. 3C is a graph showing the cell concentration of I. galbana over the course of 9 days as a monoculture.

[0028] FIGS. 4A-B demonstrate the distribution of bromoform in the whole cell culture (cells with surrounding medium), as well as separate supernatant, and cell pellet fractions from N. pellucida monoculture and N pellucida / N. sp. 2526 and N pellucida / I. galbana co-cultures. FIG. 4A is a bar graph showing the bromoform content per cell of N. pellucida mono- and cocultures on day 9 as measured in the whole cell, supernatant, and cell pellet as quantified by as quantified using GC / MS from hexane-extracted material. FIG. 4B is the same data as FIG. 4A but with a split y-axis to show the relatively small amount of bromoform that is distributed to the cell pellet, and that the relative amounts of bromoform in the N pellucida monoculture, and N. pellucida / N. sp. 2526 and A. pellucida / I. galbana co-cultures, are similar in the cell -pellet, supernatant, and whole-cell culture.

[0029] FIGS. 5A-B shows the bromoform concentration per milliliter in the whole-cell culture of the A pellucida monoculture, and A pellucida / N. sp. 2526 and A pellucida / I. galbana cocultures. FIG. 5A is a graph showing the bromoform concentration of the whole-cell culture (ng / mL) over 9 days in in the A. pellucida monoculture, and A. pellucida / N. sp. 2526 and A. pellucida / I. galbana co-cultures. FIG. 5B is a graph showing the bromoform concentration per cell (pg / cell) over 9 days in in the A. pellucida monoculture, and A. pellucida / N. sp. 2526 and A. pellucida / I. galbana co-cultures. This shows that the A. pellucida is increasing production during the first 4 days of exposure to competitor algae, and then it declines again as the competitor alga declines. FTG. 6 show the haloperoxidase activity compared to the control, as it relates to cell concentration (i.e., haloperoxidase activity per cell) in the N. pellucida monoculture, and N. pellucida / N. sp. 2526 and N. pellucidall. galbana co-cultures over 7 days.

[0030] FIG. 7 show the estimated dibromomethane (DBM) concentration and DBM distribution in the whole cell, supernatant, and cell pellet fractions for the N. pellucida monoculture, and N. pellucida / N. sp. 2526 and N. pellucidall. galbana co-cultures. Bromoform standard was used as an estimation. FIG. 7A is a graph showing the DBM concentration in the whole-cell cultures of the N. pellucida monoculture, and N. pellucida / N. sp. 2526 and N. pellucidall. galbana cocultures over 9 days. FIG. 7B is a graph showing the distribution of DBM in the whole cell culture, as well as the supernatant, and cell pellet fractions in the N. pellucida monoculture, and N. pellucida / N. sp. 2526 and N. pellucida / I. galbana co-cultures after 9 days. No DBM was found contained within the cell-pellet fraction.

[0031] FIG. 8 shows the bromoform production rate over 20 h and loss of bromoform over 7 days in N. pellucida culture medium.

[0032] FIG. 9 is a photograph of the high-lipid Botryococcus sp. and its oily exudate.

[0033] FIG. 10 is a bar graph showing bromoform stability during storage after storing at -80°C for 1 week and 1 month.

[0034] FIG. 11 is a graph of an extended stability test in culture medium (supernatant) demonstrating that bromoform produced by N. pellucida is lost at the same rate as pure bromoform in culture medium under culture conditions (20°C, light:dark 14h:10h, and 100 pmol photon light levels

[0035] FIG. 12 is a bar graph showing the bromoform recovered from a method for removing cells from cell culture, centrifuging to obtain a supernatant, and then fdtering through a 0.2 pm fdter.

[0036] FIGS. 13A-B are a series of flow cytometry images showing cell densities of N. pellucida at low, medium, and high concentrations, mixed with Isochrysis galbana cultures (and an Isochrysis galbana growing by itself as a control, on the left). Within the circled populations, each dot represents a cell. FIG. 13A shows the Isochrysis galbana control and low concentration of N. pellucida. FIG. 13B shows the medium and high concentrations of N. pellucida.

[0037] FIG 14 is a bar graph of the amount of bromoform produced when N. pellucida is grown in co-culture with Nitzschia 2526. Isochrysis galbana and Rhodomonas salina. FTGS. 15A-B are a series of flow cytometry images showing cell densities of Nitzschia 0303 at low, medium, and high concentrations, mixed with Rhodomonas salina cultures (and an Rhodomonas salina growing by itself as a control, on the left) after 5 days. Within the circled populations, each dot represents a cell. FIG. 15A shows the Rhodomonas salina control and low concentration of Nitzschia 0303. FIG. 15B shows the medium and high concentrations of Nitzschia 0303.

[0038] FIGS. 16A-B are a series of flow cytometry images showing cell densities of Nitzschia 0303 at low, medium, and high concentrations, mixed with Rhodomonas salina cultures (and an Rhodomonas salina growing by itself as a control, on the left) after 5 days where a cell scraper was not used. Within the circled populations, each dot represents a cell. FIG. 16A shows the Rhodomonas salina control and low concentration of Nitzschia 0303. FIG. 16B shows the medium and high concentrations of Nitzschia 0303.

[0039] FIGS. 17A-B are a series of flow cytometry images showing cell densities of Nitzschia 0303 at low, medium, and high concentrations, mixed with Rhodomonas salina cultures (and an Rhodomonas salina growing by itself as a control, on the left) after 6 days where a cell scraper was used. Within the circled populations, each dot represents a cell. FIG. 17A shows the Rhodomonas salina control and low concentration of Nitzschia 0303. FIG. 17B shows the medium and high concentrations of Nitzschia 0303.

[0040] FIGS. 18A-B depict an increase in bromoform when Nitzschia pellucida 0303 is grown together with the competitor diatom Nitzschia CCMP-2526. FIG. 18A is a graph showing that the bromoform concentration (ng ml"1) increases from days 3 to 12 when N pellucida 0303 is co-cultured with N. CCMP-2526 or grown in the cell-free supernatant / exudate from N. CCMP-2526, suggesting a chemical cue may be present inducing bromoform production. FIG. 18B is a graph of the N. pellucida 0303 cell concentration ( IO3cells ml"1) and bromoform concentration (ng ml"1), showing a concomitant rise, and a subsequent lag in the drop of bromoform after Day 24.

[0041] FIGS. 19A-B illustrate the results of a supernatant removal experiment in which bromoform-rich media is removed from a diatom co-culture. FIG. 19A is a graph demonstrating that maximal bromoform production rate (ng-mf' d"1) in a N pellucida 0303 and N. CCMP-2526 co-culture is realized when approximately 60% of the cell-free medium is removed. FIG. 19B is a graph of another experiment showing that the bromoform production rate (ng ml"1d"1) in a N. pelhtcida 0303 continues to increase up to 80% cell-free medium removed after removal of the supernatant every day.

[0042] FIG. 20 is a schematic of an exemplary bioreactor apparatus. The apparatus comprises two reactors, an extractor, and a recovery vessel. The first reactor vessel provides conditions for the growth of the competitor microalgae and the accumulation of the allelochemical cue for induction of bromoform production. The first reactor is fluidly connected to the second reactor, transporting the cell-free medium with the cue. The second reactor vessel provides for growth of N. pellucida 0303 and accumulation of bromoform. The second reactor vessel is fluidly connected to the separator, transporting cell -free, bromoform-rich medium to the extractor. The extractor extracts bromoform from the cell-free medium by dripping the bromoform-rich medium through a carrier oil sitting atop an aqueous phase. The carrier oil is bubbled through the aqueous phase from the bottom. Spent medium is sent to the recovery vessel, where it is enriched with nutrients, pH rebalanced, stripped of volatiles, and mixed with carbon dioxide-enriched air. The volatiles are sent to a scrubber and the recovered medium is recycled to the first reactor.

[0043] FIG. 21A-B depict a bromoform extraction experiment in canola oil and water by bubbling the oil and using an air stone in the aqueous phase. FIG.21A is an image of the lab scale bromoform extractor setup, with 220 mL canola oil in a graduated cylinder and 700 mL aqueous phase containing bromoform in an Erlenmeyer flask. The graduated cylinder and Erlenmeyer flask are connected by polypropylene tubing, an air stone is used to bubble the aqueous phase such that the bromoform is transported and accumulates in the canola oil. FIG.

[0044] 21B is a graph illustrating that the bromoform concentration increases monotonically from near 0 to above 2000 ng / mL after approximately 4.5 h, whereas the bromoform concentration in the aqueous phase drops from about 5500 ng / mL to about 500 mL.

[0045] DETAILED DESCRIPTION

[0046] The present disclosure features compositions and related methods for the treatment of microalgae to produce a small halogenated organic compound or a plurality of small halogenated organic compounds. In an aspect, the methods feature the treatment of microalgae to increase the production of a small halogenated organic compound or a plurality of small halogenated organic compounds. These small halogenated organic compounds may be useful for reducing methane production in a microorganism or rumen community. Definitions

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0048] The following definitions are more general terms used through the present disclosure. The articles “a” and “an” refer to one or more than one (e.g., at least one) of the grammatical object of an article. By way of example, “an item” means one item or more than one item.

[0049] The term “about” is used herein to mean within the typical tolerances in the art.

[0050] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the invention.

[0051] “Acquire” or “acquiring,” as used herein, refers to obtaining possession of a value, e.g., a numerical value, or image, or a physical entity (e.g., a sample), by “directly acquiring” or “indirectly acquiring” the value or physical entity. “Directly acquiring” means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. “Indirectly acquiring” refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Directly acquiring a value or physical entity includes performing a process that includes a physical change in a physical substance or the use of a machine or device. Examples of directly acquiring a value include obtaining a sample from a human subject. Directly acquiring a value includes performing a process that uses a machine or device, e.g., mass spectrometry to acquire molecular weight information.

[0052] “Allelochemical,” as used herein, refers to an exuded chemical cue that modulates the behavior of another organism, for example, a competitor microalgae. In some embodiments, the allelochemical results in a negative effect on a competitor microalgae. In other embodiments, the allelochemical results in a positive effect on a competitor microalgae. For example, Nitzschia pellucida may release a plurality of small, halogenated organic compounds as allelochemical responsive to the growth of Nitzschia 2525 in co-culture.

[0053] “Allelopathy,” as used herein, refers to the mechanism wherein a first organism, e.g., a microalgae, produces, exudes, or secretes an allelochemical for modulating the growth or behavior of a second organism, e.g., a competitor microalgae. The term “microalgae,” as used herein, refers to microscopic unicellular algae.

[0054] Microalgae are often found in marine ecosystems and can be visualized with the aid of an optical microscope. In some embodiments, the term “microalgae” refers to cyanobacteria.

[0055] A “halogen source,” or “halogenation agent,” as those terms are used interchangeably herein, refer to an agent (e.g., a small molecule or a protein) capable of modifying an entity with a halogen, for example, with a fluorine, chlorine, bromine, or iodine atom. In an embodiment, the halogenation agent is a small molecule or salt, such as potassium bromide. In another embodiment, the halogenation agent is a protein, such as a halogenase or a haloper oxidase (e.g., a vanadium haloperoxidase).

[0056] A “whole-cell culture,” as used herein, comprises both the supernatant and the cell pellet after carrying out a separation process, e.g., centrifugation. Alternatively, the “whole cell culture” may refer to both the cells and cell-free medium combined, before separation.

[0057] The term “rumen” as used herein refers to a specialized enteric compartment found within certain animals, e.g. a ruminant animal, which carries out several digestive functions within the animal, e.g. fermentative processes.

[0058] The term “ruminant animal” as used herein refers to an animal with a specialized enteric compartment which carries out several digestive functions within the animal, e.g. fermentative processes.

[0059] The terms “rumen community,” or “rumen microbial community,” as used herein, refer to a population of microorganisms including bacteria, archaea, and protozoa that populate the digestive tract of a large animal, e.g., a ruminant animal. The rumen microbial community carries out several digestive functions within the animal, including assisting in digestion to provide key nutrition to the host animal. Exemplary organisms that make up the microbial community include archaeal methanogens, examples include, Methanobrevibacter, Methanosarcina, andMethanocorpulusum. Exemplary fermentative bacterial genera found within the rumen include Coriobacteriaceae, Fibrobacter, Ruminococcus, Butyivibrio, Streptococcus, Prevotella, Succinimonas, Selenomonas, Lachnospiraand Succinivibrio.

[0060] Selected Chemical Definitions

[0061] Definitions of specific functional groups are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987.

[0062] The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical and biological arts. Also, all publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0063] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “Ci-Ce alkyl” is intended to encompass, Ci, C2, C3, C4, C5, Ce, Ci-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0064] The compounds disclosed herein may possess one or more chiral centers and so exist in a number of stereoisomeric forms. All stereoisomers and mixtures thereof are included in the scope of the present disclosure. Racemic compounds may either be separated using preparative HPLC and a column with a chiral stationary phase or resolved to yield individual enantiomers utilizing methods known to those skilled in the art. In addition, chiral intermediate compounds may be resolved and used to prepare chiral compounds of the disclosure.

[0065] The compounds disclosed herein may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including 'll.2H (D or deuterium), and3H (T or tritium); C may be in any isotopic form, including12C,13C, and14C; O may be in any isotopic form, including16O and18O; and the like.

[0066] “Alkyl” refers to a hydrocarbon group containing one or more carbon atoms, where multiple carbon atoms if present are joined by single bonds. The alkyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups having from 1 to 24 carbon atoms (“C1-C24 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-C12 alkyl”), 1 to 10 carbon atoms (“C1-C12 alkyl”), 1 to 8 carbon atoms (“Ci-Cs alkyl”), 1 to 6 carbon atoms (“Ci-C6alkyl”), 1 to 5 carbon atoms (“C1-C5 alkyl”), 1 to 4 carbon atoms (“Ci-C4alkyl”), 1 to 3 carbon atoms (“C1-C3 alkyl”), 1 to 2 carbon atoms (“C1-C2 alkyl”), or 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-C6 alkyl”).

[0067] Examples of Ci-Ce alkyl groups include methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (Ce). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (Cs) and the like. Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0068] “Haloalkyl” refers to an alkyl, wherein one or more hydrogen atoms of the hydrocarbon group are replaced with a halogen, i.e., fluorine, chlorine, bromine, and iodine. “Fluoroalkyl” refers to an alkyl, wherein one or more hydrogen atoms of the hydrocarbon group are replaced with a fluorine. “Chloroalkyl” refers to an alkyl, wherein one or more hydrogen atoms of the hydrocarbon group are replaced with a chlorine. “Bromoalkyl” refers to an alkyl, wherein one or more hydrogen atoms of the hydrocarbon group are replaced with a bromine. “lodoalkyl” refers to an alkyl, wherein one or more hydrogen atoms of the hydrocarbon group are replaced with a iodine.

[0069] The term “halo” encompasses fluoro, chloro, bromo, and iodo.

[0070] As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C2-C24 alkenyl”). In some embodiments, an alkenyl group has 2 to 12 carbon atoms (“C2-C12 alkenyl”), 2 to 10 carbon atoms (“C2-C10 alkenyl”), 2 to 8 carbon atoms (“C2-C8 alkenyl”), 2 to 6 carbon atoms (“C2-C6 alkenyl”), 2 to 5 carbon atoms (“C2-C5 alkenyl”), 2 to 4 carbon atoms (“C2-C4 alkenyl”), 2 to 3 carbon atoms (“C2-C3 alkenyl”), or 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (Ce), and the like. Each instance of an alkenyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0071] As used herein, the term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon triple bonds (“C2-C24 alkenyl”). In some embodiments, an alkynyl group has 2 to 12 carbon atoms (“C2-C10 alkynyl”), 2 to 10 carbon atoms (“C2-C10 alkynyl”), 2 to 8 carbon atoms (“C2-C8 alkynyl”), 2 to 6 carbon atoms (“C2-C6 alkynyl”), 2 to 5 carbon atoms (“C2-C5 alkynyl”), 2 to 4 carbon atoms (“C2-C4 alkynyl”), 2 to 3 carbon atoms (“C2-C3 alkynyl”), or 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Each instance of an alkynyl group may be independently optionally substituted, z.e., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0072] As used herein, the term "heteroalkyl," refers to a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -0-CH3, and -O-CH2-CH3. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Where "heteroalkyl" is recited, followed by recitations of specific heteroalkyl groups, such as -CH2O, -NRCRD, or the like, it will be understood that the terms heteroalkyl and -CH2O or -NRCRDare not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" should not be interpreted herein as excluding specific heteroalkyl groups, such as -CH2O, -NRCRD, or the like. Each instance of a heteroalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0073] As used herein, “cycloalkyl” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-C10 cycloalkyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“Cs-Cscycloalkyl”), 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”), or 5 to 10 ring carbon atoms (“C5-C10 cycloalkyl”). A cycloalkyl group may be described as, e.g., a C4-C?-membered cycloalkyl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C&), cyclohexenyl (Ce), cyclohexadienyl (Ce), and the like. Exemplary C3-C8 cycloalkyl groups include, without limitation, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (Cs), cyclooctenyl (Cs), cubanyl (Cs), bicyclo[l.l.l]pentanyl (C5), bicyclo[2.2.2]octanyl (Cs), bicyclo[2.1.1]hexanyl (Ce), bicyclo[3.1.1]heptanyl (C7), and the like. Exemplary C3-C10 cycloalkyl groups include, without limitation, the aforementioned C3-C8 cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1 7-indenyl (C9), decahydronaphthalenyl (C10), spiro [4.5] decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated. “Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.

[0074] “Heterocyclyl” as used herein refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the nonhydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety. Each instance of heterocyclyl may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.

[0075] As used herein, “hydroxy” refers to the radical -OH.

[0076] Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, and heterocyclyl groups, as defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” cycloalkyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein that result in the formation of a stable compound. The present disclosure contemplates any and all such combinations to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

[0077] Microalgae

[0078] The present disclosure features methods for producing small, halogenated organic compounds comprising microalgae. In some embodiments, the methods feature capturing small, halogenated organic compounds. In an aspect, the methods entail increasing the production of a small, halogenated organic compound or a plurality of small, halogenated organic compounds featuring microalgae. In some embodiments, the microalga is a eukaryote. In some embodiments, the microalga is a diatom. In some embodiments, the microalga is a dinoflagellate. In some embodiments, the microalga is a prokaryote. In some embodiments, the microalga is a cyanobacterium (i.e., a blue-green alga). In some embodiments, the microalga is characterized as a phytoplankton. In some embodiments, the microalga is a microphyte. In some embodiments, the microalga is photosynthetic. In some embodiments, the microalga is capable of synthesizing a chemical, e.g., a biochemical, e.g., a small halogenated organic compound. In some embodiments, the microalga grows in fresh water. In some embodiments, the microalga grows in salt water, e.g., the ocean. In some embodiments, the microalga grows in brackish water. In some embodiments, the microalga grown in open water, i.e., the water column. In some embodiments, the microalga grows in shallow water or near the water’s surface. In some embodiments, the microalga is a benthic microalga.

[0079] In some embodiments, the microalga is a heterotrophic alga. In some embodiments, the microalga is grown autotrophically. In some embodiments, the microalga is grown heterotrophically. In some embodiments, the microalga is a heterotrophic protist.

[0080] The microalgal cell may assume any shape, e.g., spherical, ellipsoidal, cylindrical, or ovoid. The microalga may be any color, e.g., green, blue, yellow, red, or brown. The microalgal cells may organize into a microcellular structure, e.g., a filamentous structure. The microalga may be biofilm-forming. The microalgal cell may be a prolate or oblate ellipsoid. In some embodiments, the microalga is capable of producing a small, halogenated organic compound. In some embodiments, the microalga is capable of producing a haloperoxidase. In some embodiments, the microalga is capable of expressing a vanadiumdependent haloperoxidase (VHPO). In some embodiments, the microalga is selected from any of the species that naturally produces a small, halogenated organic compound. In some embodiments, the microalga naturally produces bromoform. In some embodiments, the microalga naturally produces dibromomethane.

[0081] In some embodiments, the microalga has haloperoxidase activity of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, or more relative to control as measured by a Photon Technology International (PTI) QuantaMaster fluorometer. In some embodiments, the microalga has haloperoxidase activity of 100 relative to control as measured by fluorometric analysis. In some embodiments, the microalga has haloperoxidase activity of 200 relative to control as measured by a fluorometer. In some embodiments, the microalga has haloperoxidase activity of 300 relative to control as measured by fluorometry. In some embodiments, the microalga has haloperoxidase activity of 400 relative to control as measured by fluorometry. In some embodiments, the microalga has haloperoxidase activity of 500 relative to control as measured by fluorometry. In some embodiments, the microalga has haloperoxidase activity of 600 relative to control as measured by fluorometry. In some embodiments, the microalga has haloperoxidase activity of 700 relative to control as measured by fluorometry. In some embodiments, the microalga has haloperoxidase activity of 800 relative to control as measured by fluorometry. In some embodiments, the microalga has haloperoxidase activity of 900 relative to control as measured by fluorometry. In some embodiments, the microalga has haloperoxidase activity of 1000 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 1100 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 1200 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 1300 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 1400 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 1500 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 1600 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 1700 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 1800 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 1900 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 2000 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 2100 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 2200 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 2300 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 2400 relative to control as measured by fluorometric analysis and a fluorescent probe. In some embodiments, the microalga has haloperoxidase activity of 2500 relative to control as measured by fluorometric analysis and a fluorescent probe.

[0082] In some embodiments, the microalga is allelopathic or is capable of producing an allelochemical under suitable conditions. In some embodiments, the allelochemical is a small, halogenated organic compound. In some embodiments, the allelochemical is bromoform. In some embodiments, the allelochemical is dibromomethane.

[0083] In some embodiments, the microalgal species is any species of the class Bacillariophyceae (diatoms). In some embodiments, the microalgal species is any of the species of the or der Bad liar tales. In some embodiments, the microalgal species is any species of the family Badllariaceae. In some embodiments, the microalgal species is any species of the Nitzschia genus.

[0084] In some embodiments, the microalga is selected from the following species: Nitzschia acicular is.. Nitzschia amphibia, Nitzschia angustata, Nitzschia brevissima, Nitzschia clausii, Nitzschia denticula, Nitzschia disputata, Nitzschia dissipata, Nitzschia filiform is. Nitzschia fonticola, Nitzschia frigida, Nitzschia gracilis, Nitzschia heuflerania, Nitzschia frigida, Nitzschia lacuum, Nitzschia palea, Nitzschia perminuta, Nitzschia pusilia, Nitzschia recta, Nitzschia sigma, Nitzschia sigmoidea, Nitzschia sinuata, and Nitzschia tubicola, Nitzschia laevis, Nitzschia trihaeformis, or a combination thereof. In some embodiments, the microalga is Nitzschia pellucida. In some embodiments, the microalgal strain is Nitzschia cf. pellucida DCG0303. In some embodiments, the microalgal strain is Nitzschia sp. 2526.

[0085] In some embodiments, the microalgal species is any species of haptophyte. In some embodiments, the microalgal species is any species of the subphylum Haptophytina. In some embodiments, the microalgal species is any species of the class Coccolithophyceae. In some embodiments, the microalgal species is any species of the order Isochrysidales. In some embodiments, the microalgal species is any species of the family Isochrysidacaceae. In some embodiments, the microalgal species is any species of the Isochrysis genus. In some embodiments, the microalgal strain is Nitzchia cf. pellucida DCG0303.

[0086] In some embodiments, the microalga is selected from the following species:

[0087] Isochrysis galbana, Isochrysis litoralis, Isochrysis maritima, Isochrysis nuda, and Isochrysis zhanjiangensis, or a combination thereof. In some embodiments, the microalga is Isochrysis galbana.

[0088] In some embodiments, the microalgal species is any species of the phylum Dinoflagellata, i.e., the microalga is a dinoflagellate. In some embodiments, the microalgal species is any species of the class Dinophyceae. In some embodiments, the microalgal species is any species of the class Syndinea. In some embodiments, the microalgal species is any species of the class Syndiniophyceae.

[0089] In an aspect, the microalgal species is an amoeboflagellate. In some embodiments, the microalgal species is any species of the class Chlorarachniophyceae. In some embodiments, the microalgal species is any species of the order Chlorarachniales. In some embodiments, the microalgal species is any species of the family Chlorarachniaceae. In some embodiments, the microalgal species is any species of the genus Bigelowiella. In some embodiments, the microalgal species is Bigelowiella longifila.

[0090] In some embodiments, the microalga is a cyanobacterium. Cyanobacteria are prokaryotic oxyphotoautotrophs able to convert CO2 and inorganic sources of nitrogen, phosphorus, and microelements into biomass. In some embodiments, the microalga is a blue-green alga. In some embodiments, the microalgal species is any species of the class Cyanophyceae. In some embodiments, the microalgal strain is selected from any of the strains listed in Table 1 below:

[0091]

[0092] Table 1. Exemplary microalgal strains

[0093] In some embodiments, the microalgal strain has a detectable amount of a small, halogenated organic compound. In some embodiments, the small, halogenated organic compound is detectable by PT GC / MS. In some embodiments, the small, halogenated organic compound is detectable by high-throughput PT GC / MS. In some embodiments, the small, halogenated organic compound comprises bromoform.

[0094] In some embodiments, the microalgae strain is any strain selected from the strains listed in Table 2 below:

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Table 2. Exemplary microalgal strains having a detectable amount of a small, halogenated organic compound measured by high-throughput PT GC / MS.

[0108] In some embodiments, the microalgal strain is any strain selected from Bigelowiella longifila 242, Symbiodinium pseudominutum 2470, and unid. sp. 1478. In some embodiments, the microalgal strain is any strain selected from Amphidinium carterae 119, Rhodomonas sp. 767, Vitrella brassiaform 3346, Symbiodinium sp 2456, E. huxlei 3750, Ochromonas sp. 590, Symbiodinium sp. 2429, Symbiodinium sp. 2457, Ochromonas sp. 2951, Chrysoreinhard 2950, Odontella 2149, Chromera velia 2878, Chloropicon 2111, Rhizochromulina 1153, Scripsiella sp. 1073, Roseofilum eptotaenium 3313, Karenia brevis 2281, Odontella rhomus 3357, Odontella aurita 816, Chatonella marina 2962, Chatonella marina 2963, Ditylium brightwellii 361, Fibrocapsa japonica 2965, Rhodomonas sp. 768, Roseofilum eptotaenium 3575, Rhodomonas sp. 760, Skeletonema tropicum 778, Chaetoceros socialis 203, Pyramimona parkeae 725, Amorphochlo amoebiformii 3467, Ochromonas sp. 1148, Chrysoreinhard sp. 3193, Vitreliella brassicaform 3156, Symbiodinium tridacnidoru 832, Storeatula sp. 1868, Chattonella sp. 2964, Symbiodinium sp. 2592, Chrysoreinhard sp. 3202, Rhodomonas salina 1171, Heterocapsa pygmaea 1490, Chaetoceros socialis 3263, Andersenia nodulose 3482, Lingoludinium polyedra 2021, Phaeomonas parva 2877, Cyclotella baltica 3692, Isochrysis cf sp 2164, Prorocentrum cordatum 2811, Micromonas bravo 1646, Scripsiella trochoidea 3099, Amphidinium massartii 1684, Dunaliella sp. 3501, Rhodomonas abbreviate 1178, Bigelowiella longifda 242, Madagascaria erythrocladiodes 3249, Oniotrichops renformis 3252, Heterosigma akashiwo 2425, Prochlorococus marinus 1426, Chaetoceros muelleri 3754, Chrysolina cf sp 1406, Seadochloris wilhelmii 3540, Aureococcus anaphageffer 1707, Aureococcus anaphageffer 1706, Thalassiosira rotula 3703, Nannochloropsis limnetica 2260, Porphyra pulchella 3230, Heterosigma akashiwo 1870, Prorocentrum cordatum 1529, Alexandrium tamarense 2023, Alexandrium tamarense 115, Protodinium sp. 419, Proteomonas sulcata 1175, Unid sp. 1179, Skeletonema tropicum 2800, Skeletonema tropicum 2802, Poterioochro malhamensis 3181, Synechococcoys bacillaris 1333, Synechococcus sp. 3579, Symbiodinium microadriatic 2467, Rhizochromulina 1480, unid sp.1474, unid sp. 1478, Prorocentrum sp. 3721, Cylindrotheca closterium 3710 Symbiodinium goreaui 2466, Synechococcus sp. 840, Synechococcus sp. 3578, Symbiodinium kawagutii 2468, Rhizochromulina 237, Symbiodinium pilosum2 6, Madagascaria erythrocladiodes 3234, Tetraselmis sp. 932, Nannochloropsis sp. 531, Bigelowiella longifda 1485, Pingulococcus pyrenoidosis 2188, Thalassiosira diporocyclus 3732, Synechococcus sp. 3579, Synechococcus sp. 838, Skeletonema tropicum 3697, Bangiopsis subsimplex 3253, Biddulphia tridens 3574, Biddulphia tridens 3575, Lingulodiniu polyedra 2021, Brandtodinium nutriculum 3427, Porphyra lucasii 3250, Prorocentrum compressum 1786, Eunotogramma lunatum 3699, Proteomonas sp. 2715, Andersenia sp. 3436, Phormidium breve 2591, Bangiopsis subsimplex 3231, Aureococcus anaphageffer 1708, Pelagomonas calceolate 3743, Rhodomonas sp.

[0109] (duplicate) 768, Chaetoceros radicans 3706, Rhodomonas sp. 275, Pelagomonas calceolate 3738, Pelagomonas calceolate 1954, Lithmodesmium intricatum 3698, Skeletonema grenthae 3688, Phaeocystis globosa 627, Micromonas bravo 1646, Rhodomonas salina 1170, Proteomonas sulcata 704, unid sp. 1483, unid sp. 1477, Symbiodinium microadriatic 2464, Symbiodinium necroappete 2469, Rhizochromulina 1152, Prorocentrum sp. 3725, Emiliania huxleyi 3748, Entomoneis sp. 3712, Prorocentrum sp. 3722, Thalassiosira diporocyclus 3729, Amphidinium gibbosum 120, Amorphochlo amoebiformii 3469, unid sp. 1186, Thalassiosira pacifica 3885, Heterosigma akashiwo 1680, Alexandrium tamarense 1771, Karlodinium micrum 1975, Thalassiosira delicatula 3687, Heterosigma akashiwo 2393, Adenoides eludens 2081, Thecadinium yashimaense 2667, Thalassiosira weissflogii 1336, Pelagodinium beii 3395, Chaetoceros radicans 3706, Levanderina fissa 1737, Tingulodinium polyedra 1738, Cyclotella cryptica 332, Melkoniania moestrupii 3351, Heterocapsa triquetra 448, Porosira pertaporula 3680, Thalassiosira exigua 3682, Porphyridium cruentum 1328, Nannochloropsis oceanica 1779, Karlodinium micrum 1975, Prorocentrum triestinum 700, Fibrocapsa japonica 1661, Consinodiscus sp 3679, Prorocentrum cordatum 698, Karlodinium micrumn 415, Rhodosorus marinus 769, Florenciella parvula 3389, Thalassiosira gravida 3686, Cyanophora paradoxa 329, Gymnodinium fuscum 1677, Spumella sp. 3059, Thecadinium yashimaense 2726, Nephroselmi pyriformis 717, Microchlorop gaditana 526, Skeletonema marinoi 1332, Prorocentrum minimum 697, Coolia monotis 2582, Synechococcus sp. 1334, Synechococcus sp. 833, Symbiodinium trenchii 3428, Symbiodinium pseudominum 3450, Prorocentrum steidingerae 687, Pyrocystis fusiformis 3106, Symbiodinium sp. 2434, Symbiodinium dacnidorum 827, Purpureofdum apyrenoidigerum 3233, Karenia brevis 2281, Symbiodinium microadriaticu 2458, Prochlorococcus marinus 1776, Lingulodinium polyedra 407, Amphidinium gibbosum 2973, Synechococcus sp. 837, Symbiodinium antillogorgium 3449, Symbiodinium tridacnidorum 831, Symbiodinium sp. 2431, Chrysocystis fragilis 3189, Lingulodinium polyedra 1976, Alexandrium tamarense 115, Protoceratium reticulatum 3241, Phaeophila kylinii 3651, Phaeophila tenuifila 3649, Chrysocystis fragilis 3191, Symbiodinium pseudominutum 2470, Symbiodinium tridacnidorum 2465, Synechococcus sp. 836, Symbiodinium tridacnidorum 2430, Synechococcus bacillaris 1333, Symbiodinium breviolum 3573, Symbiodinium trenchii 3428, unid. sp. 1479, Protoceratium reticulatum 3241, Viridilobus marinus 3321, Thalassiothrix sp. 3718, Prorocentrum lima 684, unid. sp. 1478, Akashiwo sanguinea 3237, Karenia brevis 2229, Spumella sp. 3167, Tribonema regulare 3215, unid. sp. 3212, Prorocentrum lima 6136, Gonyaulax spinifera cf. 409, Alexandrium affine 112, Alexandrium fundyense 1719, Levanderina fissa 432, Katodinium rotundatum 1542, Alexandrium ostenfeldii 3248, Tribonema vulgare 3214, Chrysophaerella sp. 3211, Euglena deses 2910, Gonyaulax spinifera cf. 409, Scrippsiella trochoidea 1331, Alexandrium andersonii 1718, Nannochlorop oculate 2195, Protoceratium reticulatum 1889, Synura spinosa cf 3217, Mallomonas kalinae 3213, Adenoides eludens 1891, Alexandrium ostenfeldii 1773, Suriella sp. 2912, unid. sp. 2732, Synura spinosa c 3216, Thecadinium yashimaense 1890, Alexandrium lusitanicum 1888, Microchloropsis salina 1776, Alexandrium sp. 2722, Heterosigma akashiwo 3374, unid. sp. 2915, unid. sp. 2913, and Borghiella andersenii 2918.

[0110] In some embodiments, the microalgae strain is any strain selected from Nitzschia laevis 1092, Nitzschia frustrulum 558, Ditylium brightwellii 358, Ditylium brightwellii HB-G3, Ditylium brightwellii HB-B3, Ditylium brightwellii HB-B9, Ditylium brightwellii HB-G2, Nitzschia sp. 2144, Nitzschia sp. 2177, Isochrysis galbana 1323, H. rotundata HB-B9, Nitzschia sp. 2526, Rhodomonas salina 1319, and Botryococcus sp. 2724. In some embodiments, the microalgal strain is any strain selected from Nitzschia laevis 1092, Ditylium brightwellii HB-G3, Isochrysis galbana 1323, H. rotundata HB-B9, and Rhodomonas salina 1319.

[0111] In some embodiments, the microalga contains a high lipid content. In some embodiments, the high-lipid microalga produces or is capable of producing monounsaturated fatty acids and polyunsaturated fatty acids such as cn-fatty acids, e.g., co-3 fatty acids, e.g., docosahexaenoic acid (DHA) and eicosapentaenoic acid (EP A). In some embodiments, the lipid content is greater than 10%, 25%, 50%, 75% or more of the total biomass of the microalga. In some embodiments, the lipid content is 10% or more of the total biomass of the microalga. In some embodiments, the lipid content is 25% or more of the total biomass of the microalga. In some embodiments, the lipid content is 50% or more of the total biomass of the microalga. In some embodiments, the lipid content is 75% or more of the total biomass of the microalga. Exemplary high-lipid microalga include Botryococcus braunii, Isochrysis galbana, Neochloris oleoabiindans, Phaeodactyhim tricornutum, Pleurochrysis carterae, Prymnesium parvum, Tetradesmus dimorphus, Tetraselmis un. Tetraselmis suecica, and Tisochrysis lutea. In some embodiments, the high-lipid microalga is Isochrysis galbana.

[0112] In some embodiments, the present disclosure features methods for acquiring information about the presence of a small halogenated organic compound in a microalgae strain, e.g., in any of the microalgae strains listed in Table 1 above, e.g., by evaluating using GC / MS, e.g., a purge and trap GC / MS. In some embodiments, the limit of detection (LOD) is about 0.05 ng / ml for the small, halogenated organic compound, e.g., bromoform. In some embodiments, the LOD is about 0.5 ng / ml for the small, halogenated organic compound.

[0113] Allelopathy

[0114] In an aspect, the present disclosure features methods for producing small, halogenated organic compounds employing allelopathic microalgae. In some embodiments, the methods comprise culturing a plurality of microalgae, wherein one or more microalga modulates the growth or survival of another microalga by secreting an allelochemical. In some embodiments, the allelochemical is a small, halogenated organic compound, e.g., bromoform or dibromomethane. In some embodiments, the allelochemical is bromoform. In some embodiments, the allelochemical is dibromomethane.

[0115] In some embodiments, the allelochemical comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone. In an embodiment, the small, halogenated organic compound comprises 1, 1 -dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1, 3 -tribromoacetone, or 1,1,1 -tribromoacetone. In an embodiment, the small, halogenated compound comprises dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane. In an embodiment, the small, halogenated organic compound comprises 1,1 -dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1, 3 -tribromoacetone, or 1, 1, 1 -tribromoacetone, dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane.

[0116] is In some embodiments, the allelochemical is secreted from a cell into the environment. In some embodiments, the allelochemical is enriched in the supernatant relative to the cell fraction after separation, e.g., by centrifugation. The microalga may be a biofilm-forming microalga or is capable of forming a biofilm under suitable conditions. The microalga may form a biofilm when it is co-cultured with another organism, e.g., a bacterium, fungus, or a second microalga. The biofilm-forming microalga may secrete an allelochemical responsive to co-culturing with a second microalga, thereby modulating the growth or survival of the second microalga. In some embodiments, the biofilm forming microalga is induced to secrete an allelochemical responsive to co-culturing with a second microalga, thereby modulating the growth or survival of the second microalga. In some embodiments, the allelochemical is a small, halogenated organic compound. In some embodiments, the allelochemical is bromoform. In some embodiments, the allelochemical is dibromomethane. Exemplary biofilm-forming microalgae include diatoms and cyanobacteria.

[0117] In some embodiments, a first, biofilm-forming microalgae is co-cultured with a second microalgae capable of producing a small, halogenated organic compound, wherein the growth of the second microalgae is not reduced relative to the growth of the second microalgae as a monoculture. In some embodiments, a first, biofilm-forming microalgae is co-cultured with a second microalgae capable of producing a small, halogenated organic compound, wherein the strength of the biofilm of the first, biofilm-forming microalgae is greater relative to the strength of the biofilm of the first, biofilm-forming microalgae as a monoculture. In some embodiments, the first, biofilm-forming microalgae is Nitzschia spp. In some embodiments, the second microalgae is Rhodomonas salina.

[0118] In some embodiments, the microalga is characterized as rapidly growing relative to control. In some embodiments, the microalga is characterized as having reduced biomass relative to control. In some embodiments, the microalga is characterized as having high volume relative to control. In some embodiments, the microalga is characterized as achieving optimal growth in cold water. In some embodiments, the microalga is characterized as biofilm-forming and having reduced biomass relative to a control. In some embodiments, the microalga is characterized as rapidly growing and having high volume relative to control. In some embodiments, the microalga has haloperoxidase activity and is characterized as achieving optimal growth in cold water. In some embodiments, the microalga has haloperoxidase activity and is characterized as biofilm-forming and having reduced biomass relative to a control. In some embodiments, the microalga has haloperoxidase activity and is characterized as rapidly growing and having high volume relative to control. In some embodiments, the microalga having haloperoxidase activity and characterized by having optimal growth in cold water is Porosira glacialis. In some embodiments, the biofilm-forming microalga having haloperoxidase activity is Nitzschia cf. pellucida. In some embodiments, the microalga having haloperoxidase activity and characterized as rapidly growing and having high volume is Porphyridium cruentum. In some embodiments, the microalga having haloperoxidase activity and characterized as rapidly growing and having high volume is Phaeodactylum tricornutum.

[0119] Culturing Microalgae

[0120] The present disclosure features methods for producing a small, halogenated organic compound, wherein the microalga is provided in a cell culture. A cell culture comprises one or more cells in a cell culture medium. The cell culture medium can be an aqueous cell culture medium comprising components that support cell maintenance, cell viability, cell growth, and / or cell proliferation. The cell culture medium may be suitable for the production of a chemical compound, e.g., a halogenated organic compound. Exemplary media for culturing microalgae include saltwater media enriched with one or more of various salts, buffers, trace metals, amino acids, vitamins, cofactors, antibiotics, organic compounds, and saccharides, inter alia. In an embodiment, the cell culture medium is Bristol’s medium or Bristol’s modified medium. In an embodiment, the cell culture medium is Pringsheim’s medium. In an embodiment, the cell culture medium is Timothy Hay Medium.

[0121] The cell culture medium may be LI Medium, a standardized saltwater medium optimized for culturing algae. In an embodiment, the LI medium comprises a nitrate salt, a monobasic phosphate salt or hydrate thereof, a silicate salt or hydrate thereof, a trace element solution, and a vitamin solution. In an embodiment, the trace element solution comprises a chelating agent, e.g., ethylenediaminetetraacetic acid (EDTA) or a hydrate thereof, an iron salt or hydrate thereof, a manganese salt or hydrate thereof, a zinc salt or hydrate thereof, a cobalt salt or hydrate thereof, a copper salt or hydrate thereof, a molybdenum salt or hydrate thereof, a selenium salt or hydrate thereof, a nickel salt or a hydrate thereof, a vanadium salt or hydrate thereof, and chromium salt or hydrate thereof. In an embodiment, the vitamin solution comprises vitamin Bi, e.g., thiamine hydrochloride, biotin, and vitamin B12, e.g., cyanocobalamin.

[0122] LI medium may comprise about 8.82*10’4M NaNCh, 3.62*10'5M NaH2PC>4 H2O, and 1.06 *10'4M Na2SiC>3 H2O. In an embodiment, the trace element solution comprises about 1.17 * 1 O'5M Na2EDT A • 2H2O, 1.17 * 1 O'5M FeCl3• 6H2O, 9.00 * 1 O'7M MnCl2• 4H2O, 8.00 * 1 O'8M ZnSO47H2O, 5.00*10‘8M COC126H2O, 1.00*10’8M CUSO45H2O, 8.22*10’8M

[0123] Na2MoO42H2O, 1.00*10’8M H2SeO3, 1.00*10-8M NiSO4-6H2O, 1.00*10’8M Na3VO4, and 1.00*10‘8M K2CrO4. In an embodiment, the vitamin solution comprises 2.96* 10‘7M thiamine hydrochloride, 2.05*10’9M biotin, and 3.69*1O'10M cyanocobalamin.

[0124] The cell culture medium may be Prov50 medium. In an embodiment, the Prov50 medium comprises a nitrate salt, an ammonium salt, a monobasic salt or hydrate thereof, a soil extract solution, a trace metal solution, and vitamin solution. In an embodiment, the soil extract solution comprises distilled water, rich organic garden soil, and NaOH. In an embodiment, the trace metal solution comprises ethylenediaminetetraacetic acid (EDTA) or a hydrate thereof, an iron salt or hydrate thereof, a manganese salt or hydrate thereof, a zinc salt or hydrate thereof, a cobalt salt or hydrate thereof, a copper salt or hydrate thereof, a molybdenum salt or hydrate thereof, a selenium salt or hydrate thereof, a nickel salt or a hydrate thereof, a vanadium salt or hydrate thereof, and chromium salt or hydrate thereof. In an embodiment, the vitamin solution comprises thiamine hydrochloride, biotin, and vitamin BI2(cyanocobalamin).

[0125] Prov50 medium may comprise about 8.82* IO'4M NaNO3, 5.00* 10'5M NH4C1, and 3.62*10‘5M NaH2PO4H2O. In an embodiment, the trace element solution comprises 1.17* 10‘5M Na2EDTA 2H2O, 1.17*10’5M FeCl3-6H2O, 9.00*10'7M MnCl2-4H2O, 8.00*10-8M ZnSO47H2O, 5.00*10'8M COC126H2O, 1.00*10’8M CUSO45H2O, 8.22*10'8M

[0126] Na2MoO42H2O, 1.00*10’8M H2SeO3, 1.00*10‘8M NiSO4-6H2O, 1.00*10’8M Na3VO4, and 1.00* I0"8M K2CrO4. In an embodiment, the vitamin solution comprises 2.96* IO"7M thiamine hydrochloride, 2.05*10-9M biotin, and 3.69*1O10M cyanocobalamin.

[0127] The microalgal culture may be contacted with an organic compound, e.g., a saccharide, e.g., a sugar, to support the growth and proliferation of the microalgae. In an embodiment, the sugar is derived from rice. The proliferation may be monitored to attain a desired level of growth, e.g., as characterized by turbidity of the cell culture or by cell count. The microalgal culture may be allowed to proliferate in a container suitable for the proliferation of microalgae.

[0128] The culture may comprise a plurality of microalgal strains. In an embodiment, the cell culture conditions are modulated to favor the growth or proliferation of a preferred microalgal strain. In an embodiment, the cell culture conditions are modulated to obtain a particular yield of a small organic compound or a particular selectivity. In an embodiment, the cell culture conditions are modulated to induce allelopathy, e.g., to induce production of an allelochemical, e.g., a small, halogenated organic compound.

[0129] In an embodiment, the container is transparent to allow light to enter the container to promote photosynthesis. The light / dark cycle per diem may be optimized as prescribed in the art. In an embodiment, the light / dark cycle is 14 h / 10 h in order to promote proliferation of the microalgae. In an embodiment, the light / dark cycle is modulated to promote production of a small, halogenated organic compound.

[0130] The microalgae may be in culture for any period of time as prescribed in the art. For example, the microalgae may be cultured for hours, e.g., 1, 2, 3, 4, 5, 6, 12, 18, or more hours, or days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more days. In an embodiment, the microalga is cultured continuously, e.g., in a bioreactor.

[0131] Containers suitable for culturing the microalgae described herein include any cell culture container known in the art. Examples of suitable containers include, but are not limited to, a cell culture flask, a roller bottle, a bioreactor, or a tank. In a preferred embodiment, the container is a bioreactor. Other cell culture conditions appropriate for maintaining cell viability or promoting cell proliferation are known in the art.

[0132] Conditions for optimizing the proliferation of microalgae and the production of small, halogenated organic compounds in include cell density, temperature, pH, ionic strength, oxygen levels, and carbon dioxide levels. The cell density of the culture may be between about 102to 109cells / mL, e.g., about 102, 103, 104, 105, 106, 107, 108or 109cell / mL. In an embodiment, the temperature of the culture is between about 10°C to 50°C, e.g., 20°C, 25°C or 37°C. In an embodiment, the pH of the culture is between about 4 to 10, e.g., about 4, 5, 6, 7, 8, 9, or 10. In an embodiment, the ionic strength of the microalgal culture is between about 0.1 mM to 4 M.

[0133] The cell culture may be stationary or may use movement to promote maintenance or proliferation. For example, the cell culture may be rolled, shaken, or agitated to enhance cell proliferation. In an embodiment, the cell culture is disposed in a bioreactor with an impeller to promote production of a small, halogenated organic compound. The cell culture conditions disclosed herein are merely exemplary and should not be construed as limiting. In an embodiment, the bioreactor has a stirrer to promote production of a small, halogenated organic compound. In an embodiment, the bioreactor does not have a stirrer or an impeller to promote production of a small, halogenated organic compound. Halogenation of Compounds

[0134] In an aspect, the present disclosure features methods for producing a small, halogenated organic compound from a microalga via a halogenation reaction, the method comprising: (i) preparing a culture of microalgae; and (ii) acquiring information about the small, halogenated organic compound.

[0135] In a further aspect, the method comprises: (i) preparing a culture of microalgae; (ii) contacting the culture with a halogen source and a small organic compound to form a mixture; (iii) mixing the mixture; and (iv) acquiring information about the small, halogenated organic compound.

[0136] In an embodiment, the microalgae produce the small organic compound for halogenation. In an embodiment, the small organic compound may be provided in a culture medium, e.g., I medium, optionally together with a halogen source to a microalgae culture.

[0137] The small organic compound can be added at a concentration range of 1-20 g / L, 1-15 g / L, 1-10 g / L, 1-5 g / L, 2-15 g / L, 2-10g / L, 2-5 g / L, 5-20 g / L, 5-15 g / L, 5-10 g / L, 4-5 g / L, 10-20 g / L or 10-15 g / L of microalgal culture. The small organic compound can be added at a concentration of 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L or 20 g / L or more, of microalgal culture. In an embodiment, the small organic compound is added to the microalgal culture at 4 g / L once per day, or 5 g / L once per day.

[0138] The small organic compound may be a naturally occurring or non-naturally occurring compound. For example, the small organic compound may comprise a natural product, a lipid, a sterol, a steroid, an amino acid, a sugar, a phlorotannin, a tannin, a lignin, or a lignin derivative. In an embodiment, the small organic compound comprises a functional group, e.g., an aldehyde, ketone, acetyl, acyl, hydroxyl, ester, ether, amine, amide, aryl, heteroaryl, heterocyclyl, or cycloalkyl group. In an embodiment, the small organic compound comprises an alkenyl or alkynyl group. In an embodiment, the small organic compound comprises an aldehyde or ketone group. In an embodiment, the small organic compound comprises an alpha-beta unsaturated ketone. In an embodiment, the small organic compound is acetone or acetylacetone.

[0139] The small organic compound contains at least 1 carbon atom, e.g., at least 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 12 carbon atoms, or more. In an embodiment, the small organic compound comprises between 1 and 10 carbon atoms, 1 and 6 carbon atoms, 2 and 10 carbon atoms, 3 and 10 carbon atoms, 4 and 10 carbon atoms, 5 and 10 carbon atoms, or 6 and 10 carbon atoms. In some embodiments, the small organic compound is saturated. In In some embodiments, the small organic compound comprises an element of unsaturation, e.g., 2, 3, 4, 5, 6, 7, 8, or more elements of unsaturation. In an embodiment, the small organic compound further comprises an oxygen atom, a nitrogen atom, a sulfur atom, or a phosphorus atom. In an embodiment, the small organic compound comprises 1, 2, 3, 4, 5, or 6 oxygen atoms. In an embodiment, the small organic compound comprises 1, 2, 3, 4, 5, or 6 nitrogen atoms. In an embodiment, the small organic compound comprises 1, 2, 3, 4, 5, or 6 sulfur atoms. In an embodiment, the small organic compound comprises 1, 2, 3, 4, 5, or 6 phosphorus atoms.

[0140] In an embodiment, the small organic compound has a molecular weight or molecular mass of between about 15 Da and 1,500 Da, e.g., between about 15 Da and 1,250 Da, between about 15 Da and about 1,000 Da, between about 15 Da and about 750 Da, between about 15 Da and about 500 Da, between about 15 Da and about 250 Da, between about 15 Da and about 100 Da, between about 25 Da and 500 Da, between about 25 Da and 100 Da, between about 50 Da and 500 Da, between about 100 Da and 250 Da, between about 100 Da and 500 Da, or between about 100 Da and 1,000 Da.

[0141] In an embodiment, the small organic compound comprises an aldehyde, ketone, acetyl, acyl, hydroxyl, ester, ether, amine, amide, aryl, heteroaryl, heterocyclyl, or cycloalkyl group. In an embodiment, the small organic compound comprises an alkenyl or alkynyl group. In an embodiment, the small organic compound comprises an aldehyde or ketone group. In an embodiment, the small organic compound comprises an alpha-beta unsaturated ketone. In an embodiment, the small organic compound is acetone or acetyl acetone. In an embodiment, the small organic compound is a natural product, a lipid, a sterol, a steroid, an amino acid, a sugar, a phlorotannin, a tannin, a lignin, or a lignin derivative.

[0142] In one aspect, the small organic compound comprises a compound of Formula (Y):

[0143]

[0144] or a salt, tautomer, or isomer thereof, wherein each of Rla, Rlb, Rlc, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, and R5cis independently hydrogen, halogen, C1-C6alkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, or heterocyclyl, wherein each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl is optionally substituted with one or more R6; R6is halogen, Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -ORA, or -NRBRC; RAis hydrogen, C1-C6alkyl, C1-C6heteroalkyl, or C2-C6alkenyl; RBand RCare each independently hydrogen, C1-C6alkyl, or C1-C6heteroalkyl; each of m and n is independently an integer between 0 and 24; and “ - ” is a single or double bond, wherein when - is a double bond, each of R2band R3bis independently absent.

[0145] In an embodiment of Formula (Y), each of Rla, Rlb, Rlc, R2a, R2b, R3a, R3b, R5a, R5b, and R5Care each independently hydrogen. In an embodiment of Formula (Y), m is selected from 0, 1, 2, or 3. In an embodiment of Formula (Y), n is selected from 0, 1, 2, or 3. In an embodiment of Formula (Y), - is a single bond. In an embodiment of Formula (Y), each of Rla, Rlb, Rlc, R2a, R2b, R3a, R3b, R5a, R5b, and R5care each independently hydrogen, each of m and n is independently selected from 0, 1, 2, or 3, and - is a single bond.

[0146] In an embodiment of Formula (Y), each of Rla, Rlb, Rlc, R5a, R5b, and R5care each independently hydrogen, and each of m and n is 0. In an embodiment of Formula (Y), each of Rla, Rlb, Rlc, R2a, R2b, R3a, R3b, R5a, R5b, and R5care each independently hydrogen, n is 0, m is 1, and - is a single bond. In an embodiment of Formula (Y), Rlais Ci alkyl; Rlb, Rlc, R5a, R5b, and R5Care each independently hydrogen, and each of m and n is 0. In an embodiment of Formula (Y), Rlais halogen (e.g., chlorine, bromine, or iodine); Rlb, Rlc, R5a, R5b, and R5care each independently hydrogen, and each of m and n is 0.

[0147] Small, Halogenated Organic Compounds

[0148] In an embodiment, the small, halogenated organic compound is brominated, chlorinated, or iodinated. In an embodiment, the small, halogenated organic compound is brominated. In an embodiment, the small, halogenated organic compound is chlorinated. In an embodiment, the small, halogenated organic compound is iodinated. In an embodiment, the small, halogenated organic compound comprises a functional group, e.g., an aldehyde, ketone, acetyl, acyl, hydroxyl, ester, ether, amine, amide, aryl, heteroaryl, heterocyclyl, or cycloalkyl group. In an embodiment, the small, halogenated organic compound comprises an alkenyl or alkynyl group. In an embodiment, the small, halogenated organic compound comprises an aldehyde or a ketone group. In an embodiment, the small, halogenated organic compound comprises an a, P-unsaturated ketone. In an embodiment, the small, halogenated organic compound comprises 1, 2, 3, 4,5, 6, 7, or 8 carbon atoms. In an embodiment, the small, halogenated organic compound comprises 1, 2, or 3 halogen atoms. In an embodiment, the small, halogenated organic compound comprises 1, 2, or 3 bromine atoms. In an embodiment, the small, halogenated organic compound comprises 1, 2, or 3 chlorine atoms. In an embodiment, the small, halogenated organic compound comprises 1, 2, or 3 iodine atoms. In an embodiment, the small, halogenated organic compound comprises an acetone moiety. In an embodiment, the small, halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone. In an embodiment, the small, halogenated organic compound comprises 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1, 3 -tribromoacetone, or 1,1,1 -tribromoacetone. In an embodiment, the small, halogenated compound comprises dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane. In an embodiment, the small, halogenated organic compound comprises 1,1 -dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1 -tribromoacetone, di chloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane.

[0149] The small, halogenated organic compound may comprise any halogen atom, such as a chlorine atom, a bromine atom, or an iodine atom. In an embodiment, the small, halogenated organic compound comprises 1, 2, 3, 4, 5, 6, or more halogen atoms. In an embodiment, the small, halogenated organic compound comprises 1, 2, 3, 4, 5, 6, or more chlorine atoms. In an embodiment, the small, halogenated organic compound comprises 1, 2, 3, 4, 5, 6, or more bromine atoms. In an embodiment, the small, halogenated organic compound comprises 1, 2, 3, 4, 5, 6, or more iodine atoms. In an embodiment, the small, halogenated organic compound comprises bromoacetone, dibromoacetone, bromopetandione, bromoform, tribromoacetone, or a variant or analog thereof. In an embodiment, the small, halogenated organic compound comprises 1,1 -dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, 1, 1, 1 -tribromoacetone, or a variant or analog thereof. In an embodiment, the small, halogenated organic compound comprises halomethanes, e.g., dihalomethanes and trihalomethanes. In an embodiment, the small, halogenated organic compound comprises haloacetones, e.g., dihaloacetones and trihaloacetones. In an embodiment, the small, halogenated organic compound comprises bromoacetone, 1, 1 -dibromoacetone, 1,1, 3 -tribromoacetone, 1,1,1-tribromoacetone, or a variant or analog thereof. In an embodiment, the small, halogenated organic compound comprises bromomethane, dibromomethane, bromoform, or a variant or analog thereof.

[0150] In another aspect, the small, halogenated organic compound comprises a compound of Formula (Z):

[0151]

[0152] or a salt, tautomer, or isomer thereof, wherein each of Rla, Rlb, Rlc, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, and R5cis independently hydrogen, halogen, C1-C6alkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, or heterocyclyl, wherein each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl is optionally substituted with one or more R6, and at least one of Rla, Rlb, Rlc, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, and R5cis independently halogen; R6is halogen, Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -ORA, or -NRBRC; RAis hydrogen, C1-C6alkyl, C1-C6heteroalkyl, or C2-C6alkenyl; RBand RCare each independently hydrogen, C1-C6alkyl, or C1-C6heteroalkyl; each of m and n is independently selected from 0, 1, 2, or 3; and “ - ” is a single or double bond, wherein when - is a double bond, each of R2band R3bis independently absent.

[0153] In an embodiment of Formula (Z), each of Rla, Rlb, and Rlcis independently halogen or hydrogen, wherein at least one of Rla, Rlb, and Rlcis halogen. In an embodiment, the halogen is selected from chlorine, bromine, or iodine. In an embodiment of Formula (Z), each of Rla, Rlb, Rlcis independently halogen or hydrogen, wherein at least two of Rla, Rlb, and Rlcis halogen. In an embodiment, the halogen is selected from two of chlorine, bromine, or iodine. In an embodiment of Formula (Z), each of Rla, Rlb, Rlcis independently halogen. In an embodiment, the halogen is selected from chlorine, bromine, or iodine. In an embodiment of Formula (Z), each of R5a, R5b, and R5cis independently halogen or hydrogen, wherein at least one of R5a, R5b, and R5Cis halogen. In an embodiment of Formula (Z), each of R5a, R5b, and R5cis independently halogen or hydrogen, wherein at least two of R5a, R5b, and R5cis halogen. In an embodiment of Formula (Z), each of R5a, R5b, and R5cis independently halogen. In an embodiment of Formula (Z), - is a single bond. In an embodiment of Formula (Z), each of m and n is independently selected from 0, 1, 2, or 3, and - is a single bond.

[0154] Acids

[0155] The present disclosure features methods for producing a small halogenated organic compound from a microalga that may comprise treatment with an acid. In some embodiments, the acid is provided in a culture medium, e.g., LI medium, to the microalgae culture. The acid may comprise an organic acid, e.g., comprising a C1-C25 alkyl, e.g., a Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, Ce alkyl, C7 alkyl, Cs alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, Ci6 alkyl, C17 alkyl, Cis alkyl, C19 alkyl, C20 alkyl, C21 alkyl, C22 alkyl, C23 alkyl, C24 alkyl, or C25 alkyl acid moiety.

[0156] In some embodiments, the organic acid comprises a C2-C25 alkenyl, e.g., a C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, Ce alkenyl, C7 alkenyl, Cs alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl, C14 alkenyl, C15 alkenyl, Ci6 alkenyl, C17 alkenyl, Cis alkenyl, C19 alkenyl, C20 alkenyl, C21 alkenyl, C22 alkenyl, C23 alkenyl, C24 alkenyl, or C25 alkenyl acid moiety.

[0157] In some embodiments, the organic acid comprises a C2-C26 alkynyl, e.g., a C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, Ce alkynyl, C7 alkynyl, Cs alkynyl, C9 alkynyl, C10 alkynyl, C11 alkynyl, C12 alkynyl, C13 alkynyl, C14 alkynyl, C15 alkynyl, Ci6 alkynyl, C17 alkynyl, Cis alkynyl, C19 alkynyl, C20 alkynyl, C21 alkynyl, C22 alkynyl, C23 alkynyl, C24 alkynyl, or C25 alkynyl acid moiety.

[0158] In some embodiments, the organic acid comprises a C1-C25 heteroalkyl, e.g., a Ci heteroalkyl, C2 heteroalkyl, C3 heteroalkyl, C4 heteroalkyl, C5 heteroalkyl, Ce heteroalkyl, C7 heteroalkyl, Cs heteroalkyl, C9 heteroalkyl, C10 heteroalkyl, C11 heteroalkyl, C12 heteroalkyl, C13 heteroalkyl, C14 heteroalkyl, C15 heteroalkyl, Cie heteroalkyl, C17 heteroalkyl, Cis heteroalkyl, C19 heteroalkyl, C20 heteroalkyl, C21 heteroalkyl, C22 heteroalkyl, C23 heteroalkyl, C24 heteroalkyl, or C25 heteroalkyl acid moiety. In some embodiments, the method for modulating the production of small, halogenated compound features a C1-C25 heteroalkyl, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized. The heteroatom(s) O, N, P, S, and Si may be placed at any position of the heteroalkyl group.

[0159] In some embodiments, the organic acid comprises a C3-C10 cycloalkyl, e.g., a C3 cycloalkyl, C4 cycloalkyl, C5 cycloalkyl, Ce cycloalkyl, C7 cycloalkyl, Cs cycloalkyl, C9 cycloalkyl, or C10 cycloalkyl acid moiety.

[0160] In some embodiments, the organic acid comprises a C3-C10 heterocyclyl, e.g., a C3 heterocyclyl, C4 heterocyclyl, C5 heterocyclyl, Ce heterocyclyl, C7 heterocyclyl, Cs heterocyclyl, C9 heterocyclyl, or C10 heterocyclyl acid. In some embodiments, the organic acid comprises a C3-C10 aryl, e.g., a C3 aryl, C4 aryl, C5 aryl, Ce aryl, C7 aryl, Cs aryl, C9 aryl, or C10 aryl acid.

[0161] In some embodiments, the organic acid comprises a C3-C10 heteroaryl, e.g., a C3 heteroaryl, C4 heteroaryl, C5 heteroaryl, Ce heteroaryl, C7 heteroaryl, Cs heteroaryl, C9 heteroaryl, or C10 heteroaryl acid.

[0162] In some embodiments, the organic acid comprises a carboxylic acid.

[0163] In some embodiments, the organic acid comprises an alkyl (e.g., heteroalkyl), cycloalkyl (e.g., heterocyclyl), alkenyl, alkynyl, or aryl (e.g., heteroaryl) moiety.

[0164] In some embodiments, the organic acid comprises a hydroxyl, an oxo, a ketone, an aldehyde, an ether, an ester, SOX, or PCX, wherein x =1, 2, 3.

[0165] In some embodiments, the organic acid is capable of undergoing a keto-enol tautomerization.

[0166] In some embodiments, the organic acid is capable of oxidizing an electrophilic halide compound to its hypohalous acid, e.g., HOF, H0C1, HOI, or HOBr.

[0167] In some embodiments, the organic acid is a Lewis acid. In some embodiments, the organic acid is a Bronsted-Lowry acid.

[0168] In some embodiments, the organic acid is a strong acid. In some embodiments, the organic acid is a weak acid. In some embodiments, the organic acid is a superacid.

[0169] In some embodiments, the method for modulating the production of a small halogenated organic compound features an organic acid, wherein the pKa of the organic acid is between about -10 and +20, about -9 and +20, about -8 and +20, about -7 and +20, about -6 and +20, about -5 and +20, about -4 and +20, about -3 and +20, about -2 and +20, about -1 and +20, about 0 and +20, about + 1 and +20, about +2 and +20, about +3 and +20, about +4 and +20, about +5 and +20, about +6 and +20, about +7 and +20, about +8 and +20, about +9 and +20, about +10 and +20, about +11 and +20, about +12 and +20, about +13 and +20, about +14 and +20, about +15 and +20, about +16 and +20, about +17 and +20, about +18 and +20, or about +19 and +20.

[0170] Halogen Source

[0171] The present disclosure features methods for modulating the production of small, halogenated organic compounds comprising a halogen source. In some embodiments, the halogen source is provided in a culture medium, e.g., LI medium, to a microalgae culture. In some embodiments, the halogen source is an organic halide salt or an inorganic halide salt. In some embodiments, the halogen source is an inorganic halide salt, wherein the metal cation is an alkali metal cation (e.g., Li+, Na+, K+), alkali earth (e.g., Mg2+, Ca2+), Mn2+, orZn2+, inter alia. In some embodiments, the halogen source is an inorganic halide salt, wherein the halide is F’, CT, Br, or T. In some embodiments, the halogen source is selected from LiF, LiCl, LiBr, Lil, NaF, NaCl, NaBr, Nal, KF, KC1, KBr, KI, MgF2, MgCl2, MgBr2, Mgl2, CaF2, CaCl2, CaBr2, Cal2, MnF2, MnCl2, MnBr2, Mnl2, ZnF2, ZnCl2, ZnBr2, and Znl2. In a preferred embodiment, the halogen source is KBr, NaCl, or KI. In a preferred embodiment, the halogen source is potassium bromide (KBr).

[0172] Enzymes

[0173] The methods described herein may further comprise use of an exogenous enzyme to facilitate production of a small halogenated organic compound. The enzyme may be a peroxidase enzyme, such as a haloperoxidase, e.g., a haloperoxidase derived from a microalga, including cyanobacteria, as described herein, or a recombinant haloperoxidase. In some embodiments, the exogenous peroxidase, e.g., haloperoxidase, is added to the culture medium, e.g., to the LI medium. A haloperoxidase is an enzyme which catalyzes the conversion of an organic compound to a halogenated organic compound in the presence of a peroxide source, e.g., hydrogen peroxide.

[0174] The exogenous peroxidase may be any peroxidase known in nature, including a haloperoxidase. In an embodiment, the haloperoxidase is a vanadium haloperoxidase (VHPO). In an embodiment, the VHPO is a vanadium chloroperoxidase (VCPO), vanadium bromoperoxidase (VBPO), or vanadium iodoperoxidase (VIPO). In an embodiment, the VHPO is a VBPO. The peroxidase may be an microalgal haloperoxidase (e.g., endogenous to a microalgae) or a recombinant haloperoxidase which the microalgae is induced to express.

[0175] The exogenous peroxidase may be derived from an organism selected from Nitzschia cf. pellucida, Isochrysis galbana, Nitzschia sp. 2526, and Porosira glacialis. Botryococcus braunii, Isochrysis galbana, Neochloris oleoabundans, Phaeodactylum tricornutum, Pleurochrysis carterae, Prymnesium parvum, Tetradesmus dimorphus, Tetraselmis chui, Tetraselmis suecica, and Tisochrysis lutea. In an embodiment, the peroxidase is derived from Nitzschia cf. pellucida. In an embodiment, the peroxidase is derived from an organism selected from Isochrysis galbana. In an embodiment, the peroxidase is derived from Botryococcus sp. In an embodiment, the peroxidase is derived from a cyanobacterium. In an embodiment, the peroxidase is derived from Acaryochloris marina, Leptolyngbya sp. Heron Island J, Halomicronema hongdechloris C2206, Algicella marina, Synechococcus sp., and Synechococcus PCC 7335. In an embodiment, the peroxidase comprises a sequence of a peroxidase described herein, e.g., a peroxidase sequence provided in Table 1.

[0176] In an embodiment, the exogenous peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase sequence selected from the list in Table 2. In an embodiment, the peroxidase is a sequence selected from any one of SEQ ID NOs. 1-50. In an embodiment, the peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase sequence selected from SEQ ID NOs: 1-5. In an embodiment, the peroxidase has an amino acid sequence selected from any one of SEQ ID NOs. 1-8.

[0177] In an embodiment, the exogenous peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of Formula (I): X1GHAX2, wherein Xi is serine or alanine, and X2 is valine or threonine. In an embodiment, the amino acid sequence of Formula (I) is selected from SGHAV, SGHAT, AGHAV, and AGHAT. In an embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of Formula (I-a): X3YG X1GHAX2, wherein Xi is serine or alanine, X2 is valine or threonine, and X3 is serine or alanine. In an embodiment, the amino acid sequence of Formula (I-a) is selected from SYGSGHAV, SYGSGHAT, AYGAGHAV, AYGAGHAT, SYGSGHAV, SYGSGHAT, AYGAGHAV, and AYGAGHAT. In an embodiment, the peroxidase (e.g., the haloperoxidase) comprises an amino acid sequence of Formula (I-b): HPX3YG X1GHAX2, wherein Xi is serine or alanine, X2 is valine or threonine, and X3 is serine or alanine. In an embodiment, the amino acid sequence of Formula (I-a) is selected from HPSYGSGHAV, HPSYGSGHAT, HPAYGAGHAV, HPAYGAGHAT, HPSYGSGHAV, HPSYGSGHAT, HPAYGAGHAV, and HPAYGAGHAT.

[0178] In an embodiment, the exogenous peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of Formula (II): X4AGVHY, wherein X4 is methionine or tryptophan. In an embodiment, the amino acid sequence of Formula (II) is selected from MAGVHY and WAGVHY. In an embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of Formula (Il-a) is X5IGRNX4AGVHY, wherein X4 is methionine or tryptophan and X5 is serine or alanine. In an embodiment, the amino acid sequence of Formula (Il-a) is selected from SIGRNMAGVHY, SIGRNWAGVHY, AIGRNMAGVHY, and AIGRNWAGVHY.

[0179] In some embodiments, the exogenous peroxidase is a haloperoxidase. In embodiments, the haloperoxidase is a vanadium haloperoxidase (VHPO). In embodiments, the vanadium haloperoxidase is a vanadium bromoperoxidase (VBPO), vanadium chloroperoxidase (VCPO), or a vanadium iodoperoxidase (VIPO).

[0180] In some embodiments, the exogenous enzyme is a combination of haloperoxidases, e.g., a bromoperoxidase and a chloroperoxidase.

[0181] Table 3. Exemplary Peroxidase Sequences

[0182]

[0183]

[0184]

[0185]

[0186] In an embodiment, the peroxidase (e.g., the VHPO) comprises the amino acid sequence of SEQ ID NO: 1. In an embodiment, the peroxidase (e.g., the VHPO) comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1. In an embodiment, the peroxidase (e.g., the VHPO) comprises the amino acid sequence of SEQ ID NO: 2. In an embodiment, the peroxidase (e.g., the VHPO) comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2. In an embodiment, the peroxidase (e.g., the VHPO) comprises the amino acid sequence of SEQ ID NO: 3. In an embodiment, the peroxidase (e.g., the VHPO) comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 3. In an embodiment, the peroxidase (e.g., the VHPO) comprises the amino acid sequence of SEQ ID NO: 1. In an embodiment, the peroxidase (e.g., the VHPO) comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1. In an embodiment, the peroxidase (e.g., the VHPO) comprises the amino acid sequence of SEQ ID NO: 4. In an embodiment, the peroxidase (e.g., the VHPO) comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 4. In an embodiment, the peroxidase (e.g., the VHPO) comprises the amino acid sequence of SEQ ID NO: 5. In an embodiment, the peroxidase (e g., the VHPO) comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 5. In an embodiment, the peroxidase (e.g., the VHPO) comprises the amino acid sequence of SEQ ID NO: 6. In an embodiment, the peroxidase (e.g., the VHPO) comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least ^ / o, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 6. In an embodiment, the peroxidase (e.g., the VHPO) comprises the amino acid sequence of SEQ ID NO: 7. In an embodiment, the peroxidase (e.g., the VHPO) comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 7. In an embodiment, the peroxidase (e.g., the VHPO) comprises the amino acid sequence of SEQ ID NO: 8. In an embodiment, the peroxidase (e g., the VHPO) comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 8.

[0187] Other Additives

[0188] The present disclosure features methods for producing small halogenated organic compounds, as well as related compositions thereof, e.g., with other additives, e.g., other additives added to the culture medium, e.g., the LI medium. In an embodiment, the additive is capable of modulating the pH which is not functioning as a buffer, e.g., an acid or a base. In an embodiment, the additive is a base. In an embodiment, the base is a Lewis base. In an embodiment, the base is a Bronsted base. In an embodiment, the base is a strong base. In an embodiment, the base is a weak base. In an embodiment, the method described herein features a base, wherein the pKb of the base is between about 0-14, between about 1-14, between about 2-14, between about 3-14, between about 4-14, between about 5-14, between about 6-14, between about 7-14, between about 8-14, between about 9-14, between about 10-14, between about 11-14, between about 12-14, or between about 13-14. In an embodiment, the method described herein for modulating the production of small, halogenated organic compounds features a base, wherein the pKb of the base is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14.

[0189] In an embodiment, the base features a hydroxyl moiety.

[0190] In an embodiment, the base is an inorganic base. In an embodiment, the base is an organic base. Exemplary bases useful in the invention include KOH, NaOH, Mg(OH)2, Ca(OH)2, KHCO3, NaHCOs, and the like. Preferred bases include NaOH. Tn an embodiment, the method described herein for modulating the production of small, halogenated organic compounds features a buffer, e.g., a combination of a weak acid and a weak base in equilibrium to maintain the pH of the mixture at a designated value or within a range of designated values. In some embodiments, the buffer maintains the pH of the halogenation reaction between about pH 4 to about pH 10, between about pH 4 to about pH 9, or between about pH 4 to about pH 8. Exemplary buffers useful in the invention include (l-benzyl-4-triazoyl) methyl amine hydrochloride (Tris HCl), sodium hydrogen phosphate (e.g., phosphate buffered saline or PBS, 2-(N-morpholino)ethanesulfonic acid hydrochloride (MES HC1), sodium hydrogen carbonate, and the like. In some embodiments, the buffer comprises citric acid or a salt, ester or hydrate thereof. In a preferred embodiment, the buffer is citrate buffer.

[0191] In an aspect, the methods described herein for modulating the production of a small, halogenated organic compound includes a salt. In an embodiment, the salt is an inorganic salt, an organic salt, or a combination thereof. In an embodiment, the salt is an inorganic salt. In an embodiment, the salt is an organic salt. In an embodiment, the salt is an alkali metal salt, an alkali earth metal salt, or a transition metal salt. In an embodiment, the salt is an alkali metal salt. In an embodiment, the salt is an alkali earth metal salt. In an embodiment, the salt is a transition metal salt. In an embodiment, the salt is a halide salt, wherein the anion is a halide selected from fluoride, bromide, chloride, or iodide. In an embodiment, the salt is an akali metal halide salt or an alkali earth metal halide salt. In an embodiment, the salt is an alkali earth metal salt.

[0192] Exemplary salts include LiF, LiCl, LiBr, Lil, NaF, NaCl, NaBr, Nal, KF, KC1, KBr, KI, MgF2, MgCl2, MgBr2, Mgl2, CaF2, CaCl2, CaBr2, Cal2, MnF2, MnCl2, MnBr2, Mnl2, ZnF2, ZnCl2, ZnBr2, and Znl2. Preferred salts include NaCl.

[0193] Bioreactors

[0194] The present disclosure further features an apparatus for culturing microalgae, as well as for evaluating, purifying, isolating, or extracting the resultant small halogenated organic compounds. In one aspect the apparatus comprises a first reactor vessel, e.g., for containing a first microalgae culture. The vessels may be connected, e.g., fluidly connected, in series or in parallel. In some embodiments, a first plurality of vessels are connected, e.g., fluidly connected in series and a second plurality of vessels are connected, e.g., fluidly connected, in parallel. The reactor may be a batch reactor, a semi-batch reactor, or a continuous reactor. In some embodiments, the reactor is a batch reactor. In some embodiments, the reactor is a semi-batch reactor. In some embodiments, the reactor is a continuous reactor. The reactor may be a stirred tank reactor (CSTR). The reactor may be a laminar flow reactor. The reactor may comprise an impeller or a stirrer, e.g., to maintain homogenous mixing and minimize dead zones. The impeller may have one or more baffles. In some embodiments, the impeller or stirrer provides for laminar flow conditions, e.g., suitable for growth of the microalgae and / or removal or addition of media, e.g., media comprising a chemical cue, e.g., a small halogenated organic compound, e.g., bromoform. The reactor may be mixed by input of gas (e.g., CCh-enriched air). The reactor may be operated at steady state or unsteady state. In some embodiments, the reactor is operated at steady state. In some embodiments, the reactor is operated at unsteady state. The reactor vessel may be a metal (e.g., metal alloy, e.g., steel), glass (e.g., borosilicate), or ceramic vessel. The reactor vessel could be a graduated cylinder, a beaker, or an Erlenmeyer flask. The reactor vessel may be cylindrical, rectangular, spherical, ellipsoidal, toroidal, prismatic, or pyramidal. The reactor vessel may be transparent or translucent. The reactor vessel may contain an inlet or an outlet, e.g., for addition or removal of a liquid (e.g., cell media), gas (e.g., CCh-enriched air) or solid (e.g., cells). The reactor may be vented (e.g., from the top, side, or bottom of the vessel) with the outside atmosphere or hermetically sealed. The extractor can have an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) for dispersing small particles, e.g., liquid droplets (e.g., of oil or water) or bubbles (e.g., air bubbles comprising a small, halogenated organic compound, e.g., bromoform).

[0195] In another aspect, the apparatus comprises two or more vessels for the growth and harvesting of a small halogenated organic compound, e.g., bromoform. The vessels can be connected, e.g., fluidly connected, e.g., with tubing, to allow fortransport of a medium (e.g., a liquid, solid, gas, or a mixture thereof) between the two or more vessels. The tubing can be of varying length and be hermetically sealed. The tubing can be comprised of a metal, or plastic (e.g., polypropylene). In some embodiments, the medium transported between two or more vessels is a supernatant, e g., cell-free medium, e.g., cell-free medium comprising a small, halogenated organic compound, e.g., bromoform. In some embodiments, the medium is an aqueous medium enriched with nutrients, stripped of volatiles, and mixed with CO₂-enriched air suitable for culturing of microalgae. In some embodiments, the medium comprises cells, e.g., microalgae cells. In some embodiments, the medium does not comprise cells. In some embodiments, the medium is aqueous or comprises an oil. In some embodiments, the medium comprises an emulsion of two or more aqueous and oil phases. In some embodiments, the medium comprises bubbles, e.g., air bubbles, e.g., air bubbles comprising volatiles, e.g., small halogenated organic compounds, e.g., bromoform.

[0196] In some embodiments, the apparatus comprises two or more reactor vessels. The second reactor vessel may, e.g., comprise a second microalgae culture. The second reactor may be a batch reactor, a semi-batch reactor, or a continuous reactor. In some embodiments, the reactor is a batch reactor. In some embodiments, the reactor is a semi-batch reactor. In some embodiments, the reactor is a continuous stirred tank reactor (CSTR). The reactor may be a laminar flow reactor. The reactor may comprise an impeller or a stirrer, e.g., to maintain homogenous mixing and minimize dead zones. The impeller may have one or more baffles. In some embodiments, the impeller or stirrer provides for laminar flow conditions, e.g., suitable for growth of the microalgae and / or removal or addition of media, e.g., media comprising a chemical cue (e.g., allelochemical), e.g., a small halogenated organic compound, e.g., bromoform. The reactor may be mixed by input of gas (e.g., CCh-enriched air). The reactor may be operated at steady state or unsteady state. In some embodiments, the reactor is operated at steady state. In some embodiments, the reactor is operated at unsteady state. The reactor vessel may be a metal (e.g., metal alloy, e.g., steel), glass (e.g., borosilicate), or ceramic vessel. The reactor vessel could be a graduated cylinder, a beaker, or an Erlenmeyer flask. The reactor vessel may be cylindrical, rectangular, spherical, ellipsoidal, toroidal, prismatic, or pyramidal. The reactor vessel may be transparent, translucent, or opaque. The reactor vessel may contain an inlet or an outlet, e.g., for addition or removal of a liquid (e.g., cell media), gas (e.g., CCh-enriched air) or solid (e.g., cells). The reactor may be vented (e.g., from the top, side, or bottom of the vessel) with the outside atmosphere or hermetically sealed. The reactor can have an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) for dispersing small particles, e.g., liquid droplets (e.g., of oil or water) or bubbles (e.g., air bubbles compromising a small, halogenated organic compound, e.g., bromoform).

[0197] In a preferred embodiment, the first reactor vessel comprises a competitor diatom species, e.g., Nitzschia CCMP-2526, which is fluidly connected with the second reactor vessel comprising Nitzschia pellucida 0303, capable of producing a small, halogenated organic compound, e.g., bromoform responsive to the natural chemical cue of the competitor.. Without being bound by theory, transport of the cell-free medium containing the chemical cue from the first reactor vessel to the second reactor vessel induces production of the small, halogenated organic compound.

[0198] In another aspect, the apparatus further comprises a separator vessel (e.g., wherein the unit operation is extraction) and a recovery vessel. In some embodiments, the apparatus comprises an extractor vessel and a recovery vessel. In some embodiments, the apparatus comprises an extractor vessel. In some embodiments, the apparatus comprises a recovery vessel. The extractor vessel extracts or harvests the small, halogenated organic compound. In some embodiments, the extractor comprises two or more aqueous and oil phases. In some embodiments, the extractor comprises two or more oil and gas phases. The extractor vessel may be a metal (e g., metal alloy, e.g., steel), glass (e.g., borosilicate), plastic, or ceramic vessel. The extractor vessel could be a graduated cylinder, a beaker, or an Erlenmeyer flask. The extractor vessel may be cylindrical, rectangular, spherical, ellipsoidal, toroidal, prismatic, or pyramidal. The extractor vessel may be transparent, translucent, or opaque. The extractor vessel may contain an inlet or an outlet, e.g., for addition or removal of a liquid (e.g., cell media), gas (e.g., volatiles, e.g., a small halogenated organic compound, e.g., bromoform, or CCh-enriched air) or solid (e.g., cells). The extractor may be vented (e.g., from the top, side, or bottom of the vessel) with the outside atmosphere or hermetically sealed. The extractor may be operated at steady state or unsteady state. In some embodiments, the extractor is operated at steady state. The extractor may comprise an impeller or a stirrer, e.g., to maintain homogenous mixing and minimize dead zones. The impeller may have one or more baffles. The impeller or stirrer may provide for laminar or turbulent flow conditions.

[0199] The extractor can have an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) for dispersing small particles, e.g., liquid droplets (e.g., of oil or water) or bubbles (e.g., air bubbles compromising a small, halogenated organic compound, e.g., bromoform). In a preferred embodiment, cell-free aqueous medium enriched with a small, halogenated organic compound is transported to extractor containing an oil phase on top and an aqueous phase below. The enriched cell-free medium is dripped from the top of the extractor vessel through the oil phase and into the bottom aqueous phase. Simultaneously, the carrier oil is bubbled through the aqueous phase and rises to oil phase due to buoyancy. The small, halogenated organic compound, e.g., bromoform is extracted from the oil phase. The apparatus may comprise a recovery vessel, e.g., for nutrient enrichment, pH correction or rebuffering, volatile compound stripping, and the like. The recovery vessel may comprise an impeller or a stirrer, e.g., to maintain homogenous mixing and minimize dead zones. The impeller may have one or more baffles. In some embodiments, the impeller or stirrer provides for laminar flow or turbulent flow conditions. The recovery vessel may be operated at steady state or unsteady state. In some embodiments, the recovery vessel is operated at steady state. In some embodiments, the recovery vessel is operated at unsteady state. The reactor vessel may be a metal (e.g., metal alloy, e.g., steel), glass (e g., borosilicate), plastic, or ceramic vessel. In lab scale, the recovery vessel could be a graduated cylinder, a beaker, or an Erlenmeyer flask. The recovery vessel may be cylindrical, rectangular, spherical, ellipsoidal, toroidal, prismatic, or pyramidal. The recovery vessel may be transparent, translucent, or opaque. The recovery vessel may contain an inlet or an outlet, e.g., for addition or removal of a liquid (e.g., recovered or spent cell media), gas (e.g., CCh-enriched air) or solid (e.g., cells). An outlet may be connected to a scrubber for removal of volatile compounds, e.g., volatile compounds. The reactor may be vented (e.g., from the top, side, or bottom of the vessel) with the outside atmosphere or hermetically sealed. The recovery vessel can have an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) for dispersing small particles, e.g., liquid droplets (e.g., of oil or water) or bubbles (e.g., air bubbles compromising a small, halogenated organic compound, e.g., bromoform). The recovery vessel may be fluidly connected to one or more reactors, e.g., with a recycle stream, e.g., comprising recovered medium suitable for culturing microalgae.

[0200] In some embodiments, the extractor comprises an aqueous phase fluidly connected with the oil phase. The aqueous phase and the oil phase can be in the same vessel or separate vessels. The aqueous phase can be enriched with the small, halogenated organic compound (e.g., bromoform). The partition coefficient in octanol / water of the small, halogenated organic compound (e.g., bromoform) may favor accumulation of the small, halogenated organic compound (e.g., bromoform) in the oil phase at equilibrium. Exemplary oils include canola (rapeseed oil), olive oil, palm oil, sunflower oil, avocado oil, and the like. In some embodiments, the aqueous phase contains an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) to induce air bubble formation. Without being bound by theory, the air bubbles are enriched in small, halogenated organic compound (e.g., bromoform) and are transported to the oil phase. The small, halogenated organic compound (e.g., bromoform) accumulates in the oil phase. The small, halogenated organic compound (e.g., bromoform) is subsequently extracted from the oil phase.

[0201] An exemplary lab scale bromoform extractor setup consists of canola oil in a graduated cylinder and an aqueous phase containing bromoform in an Erlenmeyer flask. The graduated cylinder and Erlenmeyer flask are connected by a polypropylene tubing, and an air stone is used to bubble the aqueous phase such that the bromoform-enriched gas is transported and accumulates in the canola oil.

[0202] In a preferred embodiment, the bioreactor apparatus comprises two reactors, an extractor, and a recovery vessel. The first reactor vessel provides conditions for the growth of the competitor microalgae and the accumulation of its cue for induction of bromoform production. The first reactor is fluidly connected to the second reactor, transporting the cell-free medium with the cue. The second reactor vessel provides for growth of N. pellucida 0303 and accumulation of bromoform responsive to the allelochemical induction. The second reactor vessel is fluidly connected to the separator, transporting cell-free, bromoform-rich medium to the extractor. The extractor extracts bromoform from the cell-free medium by dripping the bromoform-rich medium through a carrier oil sitting atop an aqueous phase. The carrier oil is bubbled through the aqueous phase from the bottom. Spent medium is sent to the recovery vessel, where it is enriched with nutrients, pH rebalanced, stripped of volatiles, and mixed with carbon dioxide-enriched air. The volatiles are sent to a scrubber and the recovered medium is recycled to the first reactor.

[0203] In an alternative embodiment, the small halogenated organic compound, e.g., bromoform, is purged out of the reactor vessel for harvesting into an organic phase, e.g., an oil phase, e.g., by liquid-gas extraction. In some embodiments, the purging means comprising using air or CO2-enriched air. In some embodiments, the small halogenated organic compound, e.g., bromoform, is trapped or collected in the vessel comprising the organic phase.

[0204] In some embodiments, there is no chemical cue from the reactor vessel comprising the microalgae culture to modulate the production of the small, halogenated organic compound. In some embodiments, the supernatant or the cell-free culture medium comprising bromoform does not enter the extractor. In some embodiments, the apparatus comprises three or more reactor vessels, e.g., two reactor vessels comprising a microalgae culture and a vessel comprising an organic phase, e.g., containing the small halogenated organic compound, e.g., bromoform. Methods of Using Small, Halogenated Organic Compounds

[0205] In one aspect, the present disclosure provides methods of making an animal feed comprising small, halogenated organic compounds in order to reduce methane emissions. The method of making is particularly directed to feed suitable for ruminant animals (domesticated or wild livestock) including cattle, pigs, goats, sheep, and the like. Alternatively, the animal feed may be suitable for pet animals, e.g., dogs, cats, and the like. The method of making animal feed comprises the following steps: (i) providing a microalga under conditions sufficient to produce a small, halogenated organic compound (ii) evaluating the small, halogenated organic compound produced; (iii) separating the small, halogenated organic compound, thereby creating a product enriched in the small, halogenated organic compound; (iv) combining the product enriched in the small, halogenated organic compound with animal feed ingredients, thereby making an animal feed comprising a small, halogenated organic compound. The animal feed may assume any shape such as a cube, prill, pellet or flake. The animal feed may contain natural or synthetic protein, carbohydrates and lipids. The animal feed may contain grasses, corn, molasses, wheat, cottonseed, and other grains. The animal feed may contain preservatives.

[0206] In another aspect, the present disclosure provides methods for stabilizing a small, halogenated organic compound. In some embodiments, stabilizing is accomplished by enriching an oily phase in the small, halogenated organic compound. For example, the aqueous medium of a bioreactor comprising the small, halogenated organic compound may be separated, e.g., extracted, into an oily phase, thereby enriching the oily phase in the small, halogenated organic compound.

[0207] Methods for stabilizing a small, halogenated organic compound may encompass coculturing a microalga with a high-lipid microalga (e.g., Isochrysis galbana). In some embodiments, the method for stabilizing includes co-culturing N. pellucida with a high lipid microalga (e.g., Isochrysis galbana) in a bioreactor for nine days or more; harvesting the cells; separating the cell pellet from the supernatant (e.g., by a conventional centrifugation technique known in the art); and characterizing the amount of the small, halogenated organic compound by a suitable analytical method in the centrifugation fractions (e.g., gas chromatography / mass spectrometry), thereby increasing the stability of a small, halogenated organic compound. The method may further include characterizing the cell concentration of each microalgal species (e g., by flow cytometry), haloperoxidase activity (e.g., by aminophenyl fluorescein (APF) assay), or cell viability (e.g., by LIVE / DEAD assay). In some embodiments, the method comprises increasing the stability of bromoform. In some embodiments, the method comprises increasing the stability of dibromomethane.

[0208] In some embodiments, the first microalgal species is distinguishable from the second microalgal species by a cell counting method, e.g., flow cytometry. In some embodiments, Nitzschia cf. pellucida is distinguishable from Isochrysis galbana employing flow cytometry.

[0209] In another aspect, the present disclosure provides methods for increasing the yield of a small, halogenated organic compound. Increasing the yield may entail modulating the growth conditions of a microalgae culture. For example, the method may include modulating the growth medium, cell density, light-dark cycle, temperature, relative humidity, and the like for increasing the yield of a small, halogenated organic compound from a microalga.

[0210] In another aspect, the present disclosure features a method of increasing yield by providing a microalga in combination with an oil-producing organism. For example, the method may entail co-culturing N. pellucida with a second, oil-producing microalgal species (e.g., Phaeodactylum tricomatumor Isochrysis galban ) in a bioreactor, with suitable media for nine days or more; harvesting the cells; separating the cell pellet from the supernatant (e.g., by a conventional centrifugation method known in the art); and characterizing the amount of the small, halogenated organic compound by a suitable analytical method in the centrifugation fractions (e.g., gas chromatography / mass spectrometry), thereby increasing the yield of a small, halogenated organic compound. In some embodiments, the second oil-producing species is Botryococcus spp. The method may further include characterizing the cell concentration of each microalgae species (e.g., by flow cytometry), haloperoxidase activity (e.g., by aminophenyl fluorescein (APF) assay) or cell viability (e.g., by LIVE / DEAD assay. In some embodiments, allelopathic capabilities are characterized by monitoring oxygen evolution and other proxies for primary productivity). In some embodiments, the method comprises increasing the yield of bromoform. In some embodiments, the method comprises increasing the yield of dibromomethane.

[0211] In some embodiments, the method comprises increasing the yield of 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1, 3 -tribromoacetone, or 1,1,1 -tribromoacetone. In some embodiments, the method comprises increasing the yield of dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane. In some embodiments, the method comprises increasing the yield of 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1, 3 -tribromoacetone, or 1,1,1 -tribromoacetone, dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane.

[0212] In another aspect, the present disclosure provides methods for increasing the purity of a small, halogenated organic compound. Increasing the purity may entail methods of separating or enriching a medium containing the small, halogenated organic compound. The separation process may be any separation process such as extraction (e.g., solid-liquid extraction, liquidliquid extraction), filtration, centrifugation, sedimentation, and the like. In some embodiments, the method for increasing the purity of a small, halogenated organic compound comprises the following steps: (i) providing a first medium comprising the small, halogenated organic compound suitable for separation (ii) separating the first medium comprising the small, halogenated organic compound into a second medium enriched in the small, halogenated organic compound, thereby increasing the purity of a small, halogenated organic compound. In some embodiments, the methods for increasing the purity comprises: (i) providing an aqueous supernatant comprising the small, halogenated organic compound from a first vessel; (ii) transferring the aqueous supernatant comprising the small, halogenated organic compound from the first vessel to a second vessel; (iii) dripping an oil phase through the aqueous supernatant, thereby extracting a small, halogenated organic compound into an oleophilic extractant phase.

[0213] In another aspect, the present disclosure features methods for increasing the purity by providing a microalga in combination with an oil-producing organism. In some embodiments, the method may entail N. pellucida with a second, oil-producing microalgal species (e.g., Nitzschia sp. 2526 or Isochrysis galband) in a bioreactor, with suitable media for nine days or more; harvesting the cells; separating the cell pellet from the supernatant (e.g., by a conventional centrifugation method known in the art); and characterizing the amount of the small, halogenated organic compound by a suitable analytical method in the centrifugation fractions (e.g., gas chromatography / mass spectrometry), thereby increasing the purity of a small, halogenated organic compound. In some embodiments, the method comprises increasing the purity of bromoform. In some embodiments, the method comprises increasing the purity of

[0214] dibromomethane. In some embodiments, the method comprises increasing the purity of 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1, 3 -tribromoacetone, or 1,1,1 -tribromoacetone. In some embodiments, the method comprises increasing the purity of dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane. In some embodiments, the method comprises increasing the purity of 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1, 3 -tribromoacetone, or 1,1,1 -tribromoacetone, dichloroiodomethane, di chlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane.

[0215] In an embodiment, the cell density of the microalgal culture may be between about 102to 106cells / mL, e.g., about 102, 103, 104, 105, or 106cell / mL. In an embodiment, the temperature of the microalgal culture is between about 10°C to 50°C, e.g., 20°C, 25°C or 37°C. In an embodiment, the pH of the microalgal culture is between about 4 to 10, e.g., about 4, 5, 6, 7, 8, 9, or 10.

[0216] In some embodiments, the cell concentration of an algal monoculture increases over a period of 5, 6, 7, 8, 9 days or more in a vessel, e.g., a bioreactor. In some embodiments, the cell concentration of a first algal species of an algal co-culture increases over a period of 5, 6, 7, 8, 9 days or more in a vessel, e.g., a bioreactor. In some embodiments, the cell concentration of a N. cf. pellucida monoculture increases from about 10- 103cells / mL to about 37- 103cells / mL over a period of 9 days in a vessel, e.g., a bioreactor. In some embodiments, the cell concentration of a N. cf. pellucida in a co-culture with Nitzschia 2526 increases from about 15 • 103cells / mL to about 25 103cells / mL over a period of 9 days in a vessel, e.g., a bioreactor. In some embodiments, the cell concentration of a N. cf. pellucida in a co-culture with Isochrysis galbana increases from about 12 103cells / mL to about 28 103cells / mL over a period of 9 days in a vessel, e.g., a bioreactor.

[0217] In some embodiments, the cell concentration of a first algal species when grown as monoculture increases over a period of 5, 6, 7, 8, 9 or more days in a vessel, e.g., a bioreactor. In some embodiments, the cell concentration of a first algal species grown as a co-culture with a second algal species decreases over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9 days or more. In some embodiments, the cell concentration of N. cf. pellucida grown as a co-culture with Nitzschia 0303 decreases from about 50 - 103cells / mL to about 0 cells / mL over a period of 9 days in a vessel, e g., a bioreactor. In some embodiments, the cell concentration of N. cf. pellucida grown as a co-culture with Isochrysis galbana decreases from about 425 • 103cells / mL to about 75 • 103cells / mL over a period of 9 days in a vessel, e.g., a bioreactor.

[0218] In some embodiments, the concentration of a small, halogenated organic compound, e.g., bromoform, is higher in the supernatant fractions relative to the cell pellet after centrifugation for an algal culture grown for 5, 6, 7, 8, 9 days or more in a vessel, e.g., a bioreactor. In some embodiments, the supernatant concentration of a small, halogenated organic compound, e.g., bromoform, is 0.2 pg / cell relative to about 0 pg / cell in the cell pellet of a N. cf. pellucida monoculture grown for 9 days in a vessel, e.g., a bioreactor. In some embodiments, the supernatant concentration of a small, halogenated organic compound, e.g., bromoform, is 0.45 and 0.55 pg / cell, respectively relative to about 0.1 pg / cell in the cell pellet of a / V. cf. pellucidaN' itzschia 2526 co-culture grown for 9 days in a vessel, e.g., a bioreactor. In some embodiments, the supernatant concentration of a small, halogenated organic compound, e.g., bromoform, is 0.35 pg / cell, relative to about 0.1 pg / cell in the cell pellet of a / V. cf. pellucida, Isochrysis galbana co-culture grown for 9 days in a vessel, e.g., a bioreactor.

[0219] In some embodiments, the supernatant concentration of a small, halogenated organic compound, e g., bromoform, is 0.2 pg / cell relative to about 0.001 pg / cell in the cell pellet of a / V. cf. pellucida monoculture grown for 9 days in a vessel, e.g., a bioreactor. In some embodiments, the supernatant concentration of a small, halogenated organic compound, e.g., bromoform, is 0.45 and 0.55 pg / cell, respectively, relative to about 0.005 pg / cell in the cell pellet of a / V. cf. pellucida'Nitzschia 2526 co-culture grown for 9 days in a vessel, e g., a bioreactor. In some embodiments, the supernatant concentrations of a small, halogenated organic compound, e.g., bromoform, is 0.35 pg / cell, relative to about 0.003 pg / cell in the cell pellet of a / V. cf. pellucidadsochrysis galbana co-culture grown for 9 days in a vessel, e.g., a bioreactor.

[0220] In some embodiments, the concentration of a small, halogenated organic compound, e.g., dibromomethane, is higher in the supernatant fraction relative to the cell pellet after centrifugation for an algal culture grown for 5, 6, 7, 8, 9 days or more in a vessel, e.g., a bioreactor. In some embodiments, the supernatant concentration of a small, halogenated organic compound, e.g., dibromomethane, is about 1.3 ng / ml relative to about 0 ng / mL in the cell pellet of a N. cf. pellucida monoculture grown for 9 days in a vessel, e.g., a bioreactor. In some embodiments, the supernatant concentration of a small, halogenated organic compound, e.g., dibromomethane, is 1.8 and 2 ng / mL, respectively, relative to about 0 ng / mL in the cell pellet of aN. cf. pellucida / Nitzschia 2526 co-culture grown for 9 days in a vessel, e.g., a bioreactor. In some embodiments, the supernatant concentration of a small, halogenated organic compound, e.g., dibromomethane, is 3 ng / mL, relative to about 0 ng / mL in the cell pellet of a N. cf pellucida l ochiy sis galbana co-culture grown for 9 days in a vessel, e.g., a bioreactor.

[0221] In some embodiments, the small, halogenated organic compound, e g., bromoform, concentration over time tends to a maximum value of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ng / mL or more over about 5, 6, 7, 8, 9, days or more in a vessel, e.g., a bioreactor, with an microalgae culture. In some embodiments, the bromoform concentration attains a maximum concentration of about 8 ng / mL in a N. cf. pellucida monoculture grown for 9 days in a vessel, e.g., a bioreactor. In some embodiments, the bromoform concentration attains a maximum concentration of about 13 ng / mL in aN. cf. pellucida / Nitzschia sp. 2526 co-culture grown for 9 days in a vessel, e.g., a bioreactor. In some embodiments, the bromoform concentration attains a maximum concentration of about 10 ng / mL in aN. cf. pellucida / Isochrysis galbana co-culture grown for 9 days in a vessel, e.g., a bioreactor.

[0222] In some embodiments, the small, halogenated organic compound, e.g., dibromomethane, concentration over time tends to a maximum value of about 1,2, 3, 4, 5, 6, 7, 8, 9, 10 ng / mL or more over about 5, 6, 7, 8, 9, days or more in a vessel, e.g., a bioreactor, with an microalgae culture. In some embodiments, the dibromomethane concentration attains a maximum concentration of about 1.3 ng / mL in a N. cf. pellucida monoculture grown for 9 days in a vessel, e g., a bioreactor. In some embodiments, the dibromomethane concentration attains a maximum concentration of about 2 ng / mL in a N. cf. pellucida / Nitzschia sp. 2526 co-culture grown for 9 days in a vessel, e.g., a bioreactor. In some embodiments, the dibromomethane concentration attains a maximum concentration of about 3.8 ng / mL in a N. cf. pellucida / Isochrysis galbana coculture grown for 9 days in a vessel, e.g., a bioreactor.

[0223] In some embodiments, the small, halogenated organic compound, e.g., bromoform, concentration per cell attains a maximum concentration per cell of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 pg / cell or more 2, 3, 4, 5, 6, 7, 8, 9 days or more in a vessel, e.g., a bioreactor, with a microalgal culture. In some embodiments, the bromoform concentration per cell attains a maximum concentration of about 0.5 pg / cell on day 4 in aN. cf. pellucida monoculture. In some embodiments, the bromoform concentration per cell attains a maximum concentration of about 1.8 pg / cell on day 4 in a N. cf. pellucida! Isochrysis galbana co-culture. In some embodiments, the bromoform concentration per cell attains a maximum concentration of about 4.5 pg / cell on day 4 in a N. cf. pelhicida / N. sp. 2526 co-culture.

[0224] In some embodiments, the haloperoxidase activity per cell attains a maximum activity of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 relative to control or more 2, 3, 4, 5, 6, 7, 8, 9 days or more in a vessel, e.g., a bioreactor, with a microalgal culture. In some embodiments, the haloperoxidase is a vanadium-dependent haloperoxidase (VHPO). In some embodiments, the haloperoxidase activity per cell attains a maximum activity of about 0.5 relative to control on day 4 in a 77 cf. pellucida monoculture in a vessel, e.g., a bioreactor. In some embodiments, the haloperoxidase activity per cell attains a maximum activity of about 3.5 relative to control on day 4 in aN. cf. pellucida / N sp. 2526 co-culture in a vessel, e.g., a bioreactor. In some embodiments, the haloperoxidase activity per cell attains a maximum activity of about 2 relative to control on day 4 in a N. cf. pellucidadsochrysis galbana co-culture in a vessel, e.g., a bioreactor.

[0225] The haloperoxidase activity per volume of culture increases over a period of 2, 3, 4, 5, 6, 7, 8, 9 days or more in a vessel, e.g., a bioreactor, with a microalgae culture as measured by APF assay. In some embodiments, the haloperoxidase activity per volume of culture increases to about 1,000 / mL relative to control on day 7 for a N. cf. pellucida monoculture in a vessel, e.g., a bioreactor. In some embodiments, the haloperoxidase activity per volume of culture increases to about 15,000 / mL relative to control on day 7 for aN. cf. pellucida / N. sp. 2526 co-culture in a vessel, e.g., a bioreactor. In some embodiments, the haloperoxidase activity per volume of culture increases to about 10,000 / mL relative to control on day 7 for aN. cf. pellucida! 1 sochry sis galbana co-culture in a vessel, e.g., a bioreactor.

[0226] In some embodiments, the production rate and loss of a small, halogenated organic compound, e.g., bromoform, is determined by solvent extraction and GC / MS. In some embodiments, the amount of bromoform is determined employing a separation step, e g., centrifugation. In some embodiments, the small, halogenated organic compound, e.g., bromoform concentration, does not negligibly change as measured Oh, 2, h, 5, 10 h, 15 h, 20 h, or more after centrifugation. In some embodiments, the bromoform concentration in the whole-cell culture increases in increases after 20 h. In some embodiments, the cell density in the whole-cell culture decreases. In some embodiments, the cell density in the whole-cell culture decreases from about 79,000 cells / mL to about 73,667 cells / mL. In some embodiments, the bromoform production rate is about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.3, 0.4, 0.5 ng / (mL*h) or more. In some embodiments, the production rate is about 0.168 ng / (mL*h). Tn some embodiments, the bromoform production rate is about 0.0001, 0.001, 0.002, 0.003, 0.004, 0.005 pg / cell / h. In some embodiments, the bromoform production rate is about 0.00270 pg / cell / h. In some embodiments, the initial rate of loss is about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01 pg / cell / h. In some embodiments, the 0.00424 pg / cell / h, as characterized by measuring the supernatant and cell pellet fractions on days 0, 1, 2, 3, 4, and 7-post centrifugation.

[0227] In some embodiments, there is no significant change in the amount of a small, halogenated organic compound, e.g., bromoform, from a fractionated sample derived from a microalgae culture stored at -80°C, as measured immediately, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month or more, e.g., after centrifugation into cell pellet and supernatant fractions. In some embodiments, the bromoform concentration in the whole-cell culture and supernatant fraction of a N. cf. pellucida culture as grown in a bioreactor for 9 days is about 10 ng / mL (e.g., 7, 8, 9, 10, 11 or 12 ng / mL) as measured immediately after centrifugation and 10 ng / mL (e.g., 7, 8, 9, 10, 11 or 12 ng / mL) as measured 1-week later after storage at -80°C.

[0228] In some embodiments, a high-lipid microalga, e.g., Botryococcus braunii, which is characterized by an oily exudate, e.g, oil droplets, is capable of increasing the purity and / or yield of a small, halogenated organic compound, e.g., bromoform. In some embodiments, the oily droplets become enriched in a small, halogenated organic compound, e.g., bromoform, relative to an aqueous phase, e.g., the aqueous culture medium, e.g., the supernatant after centrifugation.

[0229] In some embodiments, there is an increase in an amount of a small, halogenated organic compound, e.g., bromoform, either when the first microalga is co-cultured with the competitor microalgae or grown in the cell-free supernatant (e.g., exudate) of the competitor microalgae. In some embodiments, the first microalga is Nitzschia sp. (CCMP-2526) and the competitor algae is Nitzschia pellucida 0303 In some embodiments, the small, halogenated organic compound is bromoform. In some embodiments, the bromoform concentration in N. CCMP 2526 / N. sp. (CCMP-2526) co-culture increases by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more ng ml’1between days 3 and 12. In some embodiments, the bromoform concentration in a N. sp. (CCMP-2526)ZV. pellucida 0303 co-culture increases by about 10 ng ml'1between days 3 and 12. In some embodiments, the small, halogenated organic compound is bromoform. In some embodiments, the bromoform concentration in N. pellucida 0303 cultured in N. sp. (CCMP-2526) supernatant increases by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more ng ml"1between days 3 and 12. In some embodiments, the bromoform concentration in a TV. pellucida 0303 cultured in N. sp. (CCMP-2526) supernatant increases by about 10 ng ml"1between days 3 and 12.

[0230] In some embodiments, the concentration of the small, halogenated organic compound, e.g., bromoform, changes concomitantly with the microalgae cell concentration. In some embodiments, the concentration of the small, halogenated organic compound, e.g., bromoform, increases concomitantly with an increase in the microalgae cell concentration. In some embodiments, the concentration of the small, halogenated organic compound, e.g., bromoform, decreases concomitantly with a decrease in the microalgae cell concentration. In some embodiments, the increase in the small, halogenated organic compound, e g., bromoform, lags behind the increase in the in the microalgae cell concentration. In some embodiments, the decrease in the concentration of the small, halogenated organic compound, e.g., bromoform, lags behind the decrease in the microalgae cell concentration. In some embodiments, the cell concentration increases by about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 103cells ml"1after about 3 weeks, and the small, halogenated organic compound, e.g., bromoform, concentration increases by about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90 100, 200, 300, 400, 500, or 1000 ng ml"1after about 4 weeks. In some embodiments, the cell concentration increases by about 70 xlO3cells ml"1after about 3 weeks, and the small, halogenated organic compound, e.g., bromoform, concentration increases by about 40 ng ml"1after about 4 weeks.

[0231] In some embodiments, the small halogenated organic compound (e.g., bromoform) production rate of a microalgae culture can be modulated by removing media rich in the in the small, halogenated organic compound (e.g., bromoform). In some embodiments, the small halogenated organic compound (e.g., bromoform) production rate can be modulated by removing about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%o, 95%, 97.5%, 99% or more of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform). In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g, bromoform) occurs when about 10% of the cell-free medium comprising the small, halogenated organic compound (eg., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 20% of the cell -free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 30% of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 40% of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 50% of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound ( e.g., bromoform) occurs when about 60% of the cell-free medium comprising the small, halogenated organic compound (eg., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 70% of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e g, bromoform) occurs when about 80% of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 90% of the cell -free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 95% of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 97.5% of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the maximal production rate of the small, halogenated organic compound (e.g., bromoform) occurs when about 99% of the cell-free medium comprising the small, halogenated organic compound (e.g., bromoform) is removed. In some embodiments, the celi-free medium is removed continuously. In some embodiments, the cell free medium is removed one or more times, which may be arbitrary or periodic, e.g., daily, every other day, every three days, every four days, every five days, every six days, weekly, every other week, monthly, and the like.

[0232] Methods of Using Small, Halogenated Organic Compounds for Reducing Production of Methane

[0233] In an aspect, the present disclosure features methods for reducing production of methane in a rumen community, the method comprising: (i) preparing a culture of microalgae; (iv) separating the small halogenated organic compound from the mixture; and (v) providing the small halogenated organic compound to a rumen community under conditions sufficient to reduce the production of methane. In an embodiment, the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In an embodiment, the methane production is reduced by between 10-75%.

[0234] In a further embodiment, the present disclosure describes methods for reducing production of methane in a manure management system, the method comprising: (i) preparing a culture of microalgae; (iv) separating the small halogenated organic compound from the mixture; (v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane. In an embodiment, the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In an embodiment, wherein the methane production is reduced by between 10-75%.

[0235] The present disclosure further recites methods for preparing animal feed for reducing the production of methane in a rumen community, the method comprising: (i) preparing a culture of microalgae; (iv) separating the small halogenated organic compound from the mixture; and (v) preparing a composition comprising a small halogenated organic compound suitable for use as an animal feed, thereby preparing animal feed for reducing the production of methane in a rumen community.

[0236] In another aspect, the present disclosure features methods for reducing production of methane in a rumen community, the method comprising: (i) preparing a culture of microalgae; (ii) contacting the culture with a halogen source and a small organic compound to form a mixture; (iii) mixing the mixture; (iv) separating the small halogenated organic compound from the mixture; and (v) providing the small halogenated organic compound to a rumen community under conditions sufficient to reduce the production of methane. In an embodiment, the method further comprises acquiring a value for the level of methane prior to the contacting of step (ii). In an embodiment, the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In an embodiment, the methane production is reduced by between 10-75%.

[0237] In a further embodiment, the present disclosure describes methods for reducing production of methane in a manure management system, the method comprising: (i) preparing a culture of microalgae; (ii) contacting the culture with a halogen source and a small organic compound to form a mixture; (iii) mixing the mixture; (iv) separating the small halogenated organic compound from the mixture; (v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane. In an embodiment, the method further comprises acquiring a value for the level of methane prior to the contacting of step (ii). In an embodiment, the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In an embodiment, the methane production is reduced by between 10-75%.

[0238] The present disclosure further recites methods for preparing animal feed for reducing the production of methane in a rumen community, the method comprising: (i) preparing a culture of microalgae; (ii) contacting the culture with a halogen source and a small organic compound to form a mixture; (iii) mixing the mixture; (iv) separating the small halogenated organic compound from the mixture; and (v) preparing a composition comprising a small halogenated organic compound suitable for use as an animal feed, thereby preparing animal feed for reducing the production of methane in a rumen community.

[0239] ENUMERATED EMBODIMENTS

[0240] 1. A method for producing a small, halogenated organic compound from microalgae via a halogenation reaction, the method comprising:

[0241] (i) preparing a culture of microalgae; and

[0242] (ii) acquiring information about the small, halogenated organic compound, thereby producing a small, halogenated organic compound.

[0243] 2. A method for producing a small, halogenated organic compound from microalgae via a halogenation reaction, the method comprising:

[0244] (i) preparing a culture of microalgae;

[0245] (ii) optionally contacting the culture with a halogen source and a small organic compound to form a mixture;

[0246] (iii) optionally mixing the mixture; and

[0247] (iv) acquiring information about the small, halogenated organic compound,

[0248] thereby producing a small, halogenated organic compound.

[0249] 3. The method of any one of the preceding emobdiments, wherein the culture is a monoculture.

[0250] 4. The method of any one of the preceding embodiments, wherein the culture is a coculture, e g., the culture comprises a plurality of microalgae strains.

[0251] 5. A method for modulating the production of a small, halogenated organic compound via a halogenation reaction, the method comprising:

[0252] (i) preparing a co-culture of microalgae, thereby initiating a halogenation reaction;

[0253] (ii) acquiring information about the small, halogenated organic compound; and

[0254] (iii) extracting the small, halogenated organic compound from the co-culture, thereby producing a small, halogenated organic compound.

[0255] 6. The method of any one of the preceding embodiments, wherein the microalgae comprises a cyanobacterium, a diatom, a haptophyte, chlororachniophyte, ulvophyte or a dinoflagellate.

[0256] 7. The method of any one of the preceding embodiments, wherein the microalgae are selected from Nitzschia cf. pelhicida, Isochrysis galbana, Porosira glacial 'is, Bigelowiella longifila, Ulvella scutata, Ditylum brightwellii, Nitzschia laevis, or a combination thereof. 8. The method of any of the preceding embodiments, wherein the microalgae are capable of forming a biofdm.

[0257] 9. The method of any of the preceding embodiments, wherein the microalgae comprise a high lipid content, e.g., a lipid content of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., 10-40% lipid content).

[0258] 10. The method of any one of the preceding embodiments, wherein the microalgae comprising a high lipid content is selected from Botryococcus braunii, Isochrysis galbana, Neochloris oleoabundans, Phaeodactylum tricornutum, Pleurochrysis carterae, Prymnesium parvum, Tetradesmus dimorphus, Tetraselmis chui, Tetraselmis suecica, and Tisochrysis lutea, or a combination thereof.

[0259] 11. The method of any one of the preceding embodiments, wherein the small halogenated organic compound has a structure of Formula (Z):

[0260] or a salt, tautomer, or isomer

[0261]

[0262] R5a, R5b, and R5cis independently hydrogen, halogen, C1-C6alkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, or heterocyclyl, wherein each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl is optionally substituted with one or more R6, and at least one of Rla, Rlb, Rlc, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, and R5cis independently halogen; R6is halogen, C1-C6 alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -ORA, or -NRBRC; RAis hydrogen, C1-C6alkyl, C1-C6heteroalkyl, or C2-C6alkenyl; RBand RCare each independently hydrogen, C1-C6alkyl, or C1-C6heteroalkyl; each of m and n is independently selected from 0, 1, 2, or 3; and “ - ” is a single or double bond, wherein when - is a double bond, each of R2band R3bis independently absent. 12. The method of any one of the preceding embodiments, wherein the small halogenated organic compound is chlorinated; brominated; iodinated; chlorinated and iodinated; chlorinated and brominated; brominated and iodinated; or chlorinated, brominated, or iodinated.

[0263] 13. The method of any one of the preceding embodiments, wherein the small halogenated organic compound is brominated.

[0264] 14. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises 1, 2, or 3 halogen atoms.

[0265] 15. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises 1, 2, or 3 bromine atoms.

[0266] 16. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises an acetone moiety.

[0267] 17. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises dibromomethane, dibromoacetone, bromopentanedione, bromoform, or tribromoacetone.

[0268] 18. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises di chloroiodomethane, dibromochloromethane, 1,1-dibromoacetone, bromoacetone, dibromomethane, 3-bromo-2,4-pentanedione, bromoform, 1,3-tribromoacetone, or 1,1,1 -tribromoacetone.

[0269] 19. The method of any one of the preceding embodiments, wherein the microalgae is allelopathic or is capable of producing an allelochemical.

[0270] 20. The method of any one of the preceding embodiments, wherein the allelochemical is a small halogenated organic compound. 21. The method of any one of the preceding embodiments, the method further comprising sequestering the small halogenated organic compound with a microalgae strain.

[0271] 22. The method of any one of the preceding embodiments, wherein the sequestering comprises culturing a microalgae strain with a high lipid content, e.g., a lipid content of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., 10-40% lipid content).

[0272] 23. The method of any one of the preceding embodiments, wherein the microalgae strain with a high lipid content is Isochrysis galbana.

[0273] 24. The method of any one of the preceding embodiments, wherein the acquiring comprises:

[0274] (i) characterizing the small halogenated organic compound, e.g., by gas chromatography or mass spectrometry;

[0275] (ii) characterizing the microalgae, e.g., the cell concentration, e.g., by flow cytometry, or cell viability, e.g., by oxygen evolution and primary productivity rates, LIVE / DEAD assay; and / or

[0276] (iii) characterizing the haloperoxidase activity, e.g., by aminophenyl fluorescein (APF) assay.

[0277] 25. The method of any one of the preceding embodiments, wherein the method further comprises (v) separating the small halogenated organic compound from the mixture.

[0278] 26. The method of any one of the preceding embodiments, wherein the (v) separating comprises centrifuging the mixture, e.g., centrifuging the mixture into cell pellet and supernatant fractions.

[0279] 27. The method of any one of the preceding embodiments, wherein the (v) separating comprises extracting the small, halogenated organic compound, e.g., by solvent extraction.

[0280] 28. The method of any one of the preceding embodiments, wherein the (v) separating comprises extracting with an oil, e.g., canola oil. 29. The method of any one of the preceding embodiments, wherein the cell density of the culture is between about 102to 106cells / mL, e.g., about 102, 103, 104, 105, 106, 107, 108, or 109cell / mL.

[0281] 30. The method of any of the preceding embodiments, wherein the temperature of the culture is between about 10°C to 50°C, e.g., 20°C, 25°C or 37°C.

[0282] 31. The method of any one of the preceding embodiments, wherein the pH of the culture is between about 4 to 10, e.g., about 4, 5, 6, 7, 8, 9, or 10.

[0283] 32. A method for reducing production of methane in a rumen community, the method comprising:

[0284] (i) preparing a culture of microalgae;

[0285] (ii) contacting the culture with a halogen source and a small organic compound to form a mixture;

[0286] (iii) mixing the mixture; and

[0287] (iv) separating the small halogenated organic compound from the mixture; and

[0288] (v) providing the small halogenated organic compound to a rumen community under conditions sufficient to reduce the production of methane.

[0289] 33. The method of any one of the preceding embodiments, further comprising acquiring a value for the level of methane prior to the contacting of step (ii).

[0290] 34. The method of any one of the preceding embodiments, wherein the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.

[0291] 35. The method of any one of the preceding embodiments, wherein the methane production is reduced by between 10-75%. 36. A method for reducing production of methane in a manure management system, the method comprising:

[0292] (i) preparing a culture of microalgae;

[0293] (ii) contacting the culture with a halogen source and a small organic compound to form a mixture;

[0294] (iii) mixing the mixture;

[0295] (iv) separating the small halogenated organic compound from the mixture;

[0296] (v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane.

[0297] 37. The method of any one of the preceding embodiments, further comprising acquiring a value for the level of methane prior to the contacting of step (ii).

[0298] 38. The method of any one of the preceding embodiments, wherein the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.

[0299] 39. A method for preparing animal feed for reducing the production of methane in a rumen community, the method comprising:

[0300] (i) preparing a culture of microalgae;

[0301] (ii) contacting the culture with a halogen source and a small organic compound to form a mixture;

[0302] (iii) mixing the mixture;

[0303] (iv) separating the small halogenated organic compound from the mixture; and

[0304] (v) preparing a composition comprising a small halogenated organic compound suitable for use as an animal feed,

[0305] thereby preparing animal feed for reducing the production of methane in a rumen community.

[0306] 40. A method for reducing production of methane in a rumen community, the method comprising: (i) preparing a culture of microalgae;

[0307] (iv) separating the small halogenated organic compound from the mixture; and

[0308] (v) providing the small halogenated organic compound to a rumen community under conditions sufficient to reduce the production of methane.

[0309] 41. The method of any one of the preceding embodiments, wherein the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.

[0310] 42. The method of any one of the preceding embodiments, wherein the methane production is reduced by between 10-75%.

[0311] 43. A method for reducing production of methane in a manure management system, the method comprising:

[0312] (i) preparing a culture of microalgae;

[0313] (iv) separating the small halogenated organic compound from the mixture; and

[0314] (v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane.

[0315] 44. The method of any one of the preceding embodiments, wherein the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.

[0316] 45. A method for preparing animal feed for reducing the production of methane in a rumen community, the method comprising:

[0317] (i) preparing a culture of microalgae;

[0318] (iv) separating the small halogenated organic compound from the mixture; and

[0319] (v) preparing a composition comprising a small halogenated organic compound suitable for use as an animal feed,

[0320] thereby preparing animal feed for reducing the production of methane in a rumen community. 46. An apparatus for culturing a microalgal species, comprising a vessel for producing a small, halogenated organic compound.

[0321] 47. The apparatus of any one of the preceding embodiments, wherein the vessel is a reactor vessel, a separator vessel (e.g., an extractor), or a recovery vessel.

[0322] 48. The apparatus of any one of the preceding embodiments, wherein the extractor comprises an aqueous phase and an oily phase.

[0323] 49. The apparatus of any one of the preceding embodiments, wherein the extractor comprises a gaseous phase and an aqueous phase.

[0324] 50. The apparatus of any one of the preceding embodiments, comprising a plurality of vessels, e.g., reactor vessels.

[0325] 51. The apparatus of any one of the preceding embodiments, wherein each vessel in the plurality is fluidly connected to at least one of the vessels in the plurality.

[0326] 52. The apparatus of any one of the preceding embodiments, wherein the first reactor vessel comprises microalgae exuding a particular cue, and the second reactor vessel comprises microalgae capable of modulating production of a small, halogenated organic compound responsive to the competitor cue.

[0327] 53. An apparatus for producing a small, halogenated organic compound: the apparatus comprising a plurality of vessels:

[0328] a first reactor vessel comprising a first microalga capable of exuding an allelochemical cue, that can be detected by competitor algae;

[0329] a second reactor vessel comprising a second microalgae capable of modulating production of a small, halogenated organic compound responsive to the competitor cue;

[0330] an extractor for harvesting the small, halogenated organic compound; and a recovery vessel for nutrient enrichment, pH correction, or volatile compound stripping; and, wherein each vessel is fluidly connected to at least one of the vessels in the plurality.

[0331] 54. The apparatus of any one of the preceding embodiments, wherein the small, halogenated organic compound is bromoform.

[0332] 55. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is a batch reactor, a semi-batch reactor, or a continuous reactor, e.g., a continuous stirred-tank reactor (CSTR).

[0333] 56. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is the batch reactor.

[0334] 57. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is the semi-batch reactor.

[0335] 58. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is the continuous reactor, e.g., the continuous stirred-tank reactor (CSTR).

[0336] 59. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is operated at steady state or unsteady state.

[0337] 60. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is operated at steady state.

[0338] 61. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is operated at unsteady state.

[0339] 62. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises an impeller or a stirrer. 63. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises an impeller.

[0340] 64. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises a stirrer.

[0341] 65. The apparatus of any one of the preceding embodiments, wherein the impeller comprises one or more baffles.

[0342] 66. The apparatus of any one of the preceding embodiments, wherein the impeller or stirrer provides for laminar flow conditions, e.g., suitable for growth of the microalgae and / or removal or addition of media, e.g., media comprising a chemical cue (e.g., allelochemical), e g., a small halogenated organic compound, e.g., bromoform.

[0343] 67. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is a metal (e.g., metal alloy, e.g., steel), glass (e.g., borosilicate), plastic, or ceramic vessel.

[0344] 68. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is a metal (e.g., metal alloy, e.g., steel) vessel.

[0345] 69. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is a glass (e.g., borosilicate) vessel.

[0346] 70. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is a plastic vessel.

[0347] 71. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is a plastic vessel.

[0348] 72. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is cylindrical, rectangular, spherical, ellipsoidal, toroidal, prismatic, or pyramidal. 73. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is cylindrical.

[0349] 74. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is rectangular.

[0350] 75. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is spherical.

[0351] 76. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is ellipsoidal.

[0352] 77. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is toroidal.

[0353] 78. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is prismatic.

[0354] 79. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is pyramidal.

[0355] 80. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is transparent or translucent.

[0356] 81. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is transparent.

[0357] 82. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is translucent. 83. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises an inlet or an outlet, e.g., for addition or removal of a liquid (e.g., cell media), gas (e.g., CO2-enriched air), or solid (e.g., cells).

[0358] 84. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises an inlet for addition of a liquid (e.g., cell media).

[0359] 85. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises an inlet for addition of a gas (e.g., CCh-enriched air).

[0360] 86. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises an inlet for addition of a solid (e.g., cells).

[0361] 87. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises an outlet for removal of a liquid (e.g., cell media).

[0362] 88. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises an outlet for removal of a gas (e.g., CCh-enriched air).

[0363] 89. The apparatus of any one of the preceding embodiments, wherein the reactor vessel comprises an outlet for removal of a solid (e.g., cells).

[0364] 90. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is vented (e g., from the top, side, or bottom of the vessel, e.g., with the outside atmosphere).

[0365] 91. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is vented from the top of the vessel.

[0366] 92. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is vented from the side of the vessel. 93. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is vented from the bottom of the vessel.

[0367] 94. The apparatus of any one of the preceding embodiments, wherein the reactor vessel is hermetically sealed.

[0368] 95. The apparatus of any one of the preceding embodiments, wherein the reactor vessel further comprises an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) for dispersing small particles, e.g., liquid droplets (e.g., of oil or water) or bubbles (e.g., air bubbles compromising a small, halogenated organic compound, e.g., bromoform).

[0369] 96. The apparatus of any one of the preceding embodiments, wherein the reactor vessels are fluidly connected with tubing, thereby allowing transporting of a medium (e.g., a liquid, solid, gas, or a mixture thereof) between the two or more vessels.

[0370] 97. The apparatus of any one of the preceding embodiments, wherein the tubing is hermetically sealed.

[0371] 98. The apparatus of any one of the preceding embodiments, wherein the tubing is comprised of a metal, or plastic (e g., polypropylene).

[0372] 99. The apparatus of any one of the preceding embodiments, wherein the tubing is comprised of a metal.

[0373] 100. The apparatus of any one of the preceding embodiments, wherein the tubing is comprised of a plastic (e.g., polypropylene).

[0374] 101. The apparatus of any one of the preceding embodiments, wherein the medium comprises a supernatant, e.g., cell-free medium, e.g., cell-free medium comprising a small, halogenated organic compound, e.g., bromoform. 102. The apparatus of any one of the preceding embodiments, wherein the medium is an aqueous medium enriched with nutrients, stripped of volatiles, and mixed with CO₂-enriched air suitable for culturing of microalgae.

[0375] 103. The apparatus of any one of the preceding embodiments, wherein the medium is an aqueous medium enriched with nutrients suitable for culturing a microalgae.

[0376] 104. The apparatus of any one of the preceding embodiments, wherein the medium is an aqueous medium stripped of volatiles suitable for culturing a microalgae.

[0377] 105. The apparatus of any one of the preceding embodiments, wherein the medium is an aqueous medium mixed with CCh-enriched air for culturing a microalgae.

[0378] 106. The apparatus of any one of the preceding embodiments, wherein the medium does not comprise cells.

[0379] 107. The apparatus of any one of the preceding embodiments, wherein the medium is aqueous.

[0380] 108. The apparatus of any one of the preceding embodiments, wherein the medium comprises an oil.

[0381] 109. The apparatus of any one of the preceding embodiments, wherein the medium comprises an emulsion of two or more aqueous and oil phases.

[0382] 110. The apparatus of any one of the preceding embodiments, wherein the medium comprises an emulsion of two aqueous and oil phases.

[0383] 111. The apparatus of any one of the preceding embodiments, wherein the medium comprises bubbles, e.g., air bubbles, e.g., air bubbles comprising volatiles, e.g., small halogenated organic compounds, e.g., bromoform. 112. The apparatus of any one of the preceding embodiments, wherein the separator vessel comprises an extractor vessel.

[0384] 113. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises two or more aqueous and oil phases.

[0385] 114. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is operated at steady state or unsteady state.

[0386] 115. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is operated at steady state.

[0387] 116. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is operated at unsteady state.

[0388] 117. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an impeller or a stirrer.

[0389] 118. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an impeller.

[0390] 119. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises a stirrer.

[0391] 120. The apparatus of any one of the preceding embodiments, wherein the impeller comprises one or more baffles.

[0392] 121. The apparatus of any one of the preceding embodiments, wherein the impeller or stirrer provides for laminar flow conditions, e.g., suitable for growth of the microalgae and / or removal or addition of media, e.g., media comprising a chemical cue (e.g., allelochemical), e.g., a small halogenated organic compound, e.g., bromoform. 122. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is a metal (e.g., metal alloy, e.g., steel), glass (e.g., borosilicate), plastic or ceramic vessel.

[0393] 113. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is a metal (e.g., metal alloy, e.g., steel) vessel.

[0394] 114. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is a glass (e.g., borosilicate) vessel.

[0395] 115. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is a plastic vessel.

[0396] 116. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is cylindrical, rectangular, spherical, ellipsoidal, toroidal, prismatic, or pyramidal.

[0397] 117. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is cylindrical.

[0398] 118. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is rectangular.

[0399] 120. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is spherical.

[0400] 121. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is ellipsoidal.

[0401] 122. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is toroidal. 123. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is prismatic.

[0402] 124. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is pyramidal.

[0403] 125. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is transparent or translucent.

[0404] 126. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is transparent.

[0405] 127. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is translucent.

[0406] 128. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an inlet or an outlet, e.g., for addition or removal of a liquid (e.g., cell media), gas (e.g., CCh-enriched air), or solid (e.g., cells).

[0407] 129. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an inlet for addition of a liquid (e g., cell media).

[0408] 130. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an inlet for addition of a gas (e.g., CCh-enriched air).

[0409] 131. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an inlet for addition of a solid (e.g., CCh-cells).

[0410] 132. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an outlet for removal of a liquid (e.g., cell media). 133. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an outlet for removal of a gas (e.g., CCh-enriched air).

[0411] 134. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an outlet for removal of a solid (e.g., CCh-cells).

[0412] 135. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is vented (e g., from the top, side, or bottom of the vessel, e.g., with the outside atmosphere).

[0413] 136. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is vented from the top of the vessel.

[0414] 137. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is vented from the side of the vessel.

[0415] 138. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is vented from the bottom of the vessel.

[0416] 139. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is hermetically sealed.

[0417] 140. The apparatus of any one of the preceding embodiments, wherein the extractor vessel further comprises an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) for dispersing small particles, e.g., liquid droplets (e.g., of oil or water) or bubbles (e.g., air bubbles compromising a small, halogenated organic compound, e.g., bromoform).

[0418] 141. The apparatus of any one of the preceding embodiments, wherein the extractor vessel further comprises an atomizer.

[0419] 142. The apparatus of any one of the preceding embodiments, wherein the extractor vessel further comprises a nebulizer. 143. The apparatus of any one of the preceding embodiments, wherein the extractor vessel further comprises an aerosolizer.

[0420] 144. The apparatus of any one of the preceding embodiments, wherein the extractor vessel further comprises a bubbler (e.g., an airstone).

[0421] 145. The apparatus of any one of the preceding embodiments, wherein the extractor vessel is fluidly connected to one or more vessels in the apparatus with tubing, thereby allowing transporting of a medium (e.g., a liquid, solid, gas, or a mixture thereof) between the two or more vessels.

[0422] 146. The apparatus of any one of the preceding embodiments, wherein the tubing is hermetically sealed.

[0423] 147. The apparatus of any one of the preceding embodiments, wherein the tubing is comprised of a metal, or plastic (e.g., polypropylene).

[0424] 148. The apparatus of any one of the preceding embodiments, wherein the tubing is comprised of a metal.

[0425] 149. The apparatus of any one of the preceding embodiments, wherein the tubing is comprised of a plastic (e.g., polypropylene).

[0426] 150. The apparatus of any one of the preceding embodiments, wherein the medium comprises a supernatant, e.g., cell-free medium, e.g., cell-free medium comprising a small, halogenated organic compound, e.g., bromoform.

[0427] 151. The apparatus of any one of the preceding embodiments, wherein the medium is an aqueous medium enriched with nutrients, stripped of volatiles, and mixed with CO₂-enriched air suitable for culturing of microalgae. 152. The apparatus of any one of the preceding embodiments, wherein the medium is an aqueous medium enriched with nutrients suitable for culturing of microalgae.

[0428] 153. The apparatus of any one of the preceding embodiments, wherein the medium is an aqueous medium stripped of volatiles suitable for culturing of microalgae.

[0429] 154. The apparatus of any one of the preceding embodiments, wherein the medium is an aqueous medium mixed with CO₂-enriched air suitable for culturing of microalgae.

[0430] 155. The apparatus of any one of the preceding embodiments, wherein the medium does not comprise cells.

[0431] 156. The apparatus of any one of the preceding embodiments, wherein the medium is aqueous.

[0432] 157. The apparatus of any one of the preceding embodiments, wherein the medium comprises an oil.

[0433] 158. The apparatus of any one of the preceding embodiments, wherein the medium comprises an emulsion of two or more aqueous and oil phases.

[0434] 159. The apparatus of any one of the preceding embodiments, wherein the medium comprises an emulsion of two aqueous and oil phases.

[0435] 160. The apparatus of any one of the preceding embodiments, wherein the medium comprises bubbles, e.g., air bubbles, e.g., air bubbles comprising volatiles, e.g., small halogenated organic compounds, e.g., bromoform.

[0436] 161. The apparatus of any one of the preceding embodiments, wherein the extractor vessel comprises an aqueous phase fluidly connected with the oil phase. 162. The apparatus of any one of the preceding embodiments, wherein the aqueous phase and the oily phase are in the same vessel or separate vessels.

[0437] 163. The apparatus of any one of the preceding embodiments, wherein the aqueous phase and the oily phase are in the same vessel.

[0438] 164. The apparatus of any one of the preceding embodiments, wherein the aqueous phase and the oily phase are in separate vessels.

[0439] 165. The apparatus of any one of the preceding embodiments, wherein the aqueous phase is enriched with the small, halogenated organic compound (e.g., bromoform).

[0440] 166. The apparatus of any one of the preceding embodiments, wherein the partition coefficient in octanol / water of the small, halogenated organic compound (e.g., bromoform) favors accumulation of the small, halogenated organic compound (e.g., bromoform) in the oil phase at equilibrium.

[0441] 167. The apparatus of any one of the preceding embodiments, wherein the oil comprises canola (rapeseed) oil, olive oil, palm oil, sunflower oil, or avocado oil.

[0442] 168. The apparatus of any one of the preceding embodiments, wherein the oil comprises canola (rapeseed) oil.

[0443] 169. The apparatus of any one of the preceding embodiments, wherein the oil comprises olive oil.

[0444] 170. The apparatus of any one of the preceding embodiments, wherein the oil comprises palm oil.

[0445] 171. The apparatus of any one of the preceding embodiments, wherein the oil comprises sunflower oil. 172. The apparatus of any one of the preceding embodiments, wherein the oil comprises avocado oil.

[0446] 173. The apparatus of any one of the preceding embodiments, wherein the aqueous phase comprises an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) to induce air bubble formation.

[0447] 174. The apparatus of any one of the preceding embodiments, wherein the aqueous phase comprises an atomizer.

[0448] 175. The apparatus of any one of the preceding embodiments, wherein the aqueous phase comprises a nebulizer.

[0449] 176. The apparatus of any one of the preceding embodiments, wherein the aqueous phase comprises an aerosolizer.

[0450] 177. The apparatus of any one of the preceding embodiments, wherein the aqueous phase comprises a bubbler (e.g., an airstone).

[0451] 178. The apparatus of any one of the preceding embodiments, comprising a recovery vessel.

[0452] 179. The apparatus of any one of the preceding embodiments, wherein the recovery vessel provides for nutrient enrichment, pH correction or rebuffering, or volatile compound stripping.

[0453] 180. The apparatus of any one of the preceding embodiments, wherein the recovery vessel provides for nutrient enrichment.

[0454] 181. The apparatus of any one of the preceding embodiments, wherein the recovery vessel provides for pH correction or rebuffering. 182. The apparatus of any one of the preceding embodiments, wherein the recovery vessel provides for pH correction.

[0455] 183. The apparatus of any one of the preceding embodiments, wherein the recovery vessel provides for rebuffering.

[0456] 184. The apparatus of any one of the preceding embodiments, wherein the recovery vessel provides for volatile compound stripping.

[0457] 185. The apparatus of any one of the preceding embodiments, wherein the small, halogenated compound, e.g., bromoform, is purged out of a reactor vessel for harvesting into an organic phase, e.g., oil phase, e g., by liquid gas extraction.

[0458] 186. The apparatus of any one of the preceding embodiments, wherein an extractor vessel performs liquid-gas extraction, thereby harvesting the small, halogenated organic compound.

[0459] EXAMPLES

[0460] The present disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compositions, methods, and devices of the present disclosure and practice the claimed methods. The following working examples specifically point out various aspects of the present disclosure and are not to be construed as limiting in any way the remainder of the disclosure.

[0461] Example 1. Modulating Production of Bromoform in Microalgae Co-Culture Employing Inducible Allelopathy The example set forth below describes methods for the production of small, halogenated organic compounds, e.g., bromoform and dibromomethane, in microalgae mono- or co-culture employing inducible allelopathy. Briefly, a number of microalgal strains were screened for haloperoxidase activity relative to a cell-free culture medium control (as shown in FIG. 1) and their ability to produce small, halogenated organic compounds, e.g., bromoform and other halogenated compounds. Although Porosira glacialis had the highest intrinsic haloperoxidase activity by a significant margin, Nitzschia cf. pellucida was the only microalgal species found to demonstrably have ability to produce bromoform. N. pellucida is characterized as a biofilmforming strain that produces less biomass, and it is hypothesized that it may secrete bromoform along with other halogenated compounds, e.g., cyanogen bromide, allelopathically to modulate the encroachment of other competitive species.

[0462] As such, conditions were sought to increase the production of bromoform by culturing N pellucida as a monoculture and as a co-culture with other microalgae as follows: (i) Nitzschia pellucida, (ii) N. pellucida and Nitzschia sp. 2526, (iii) N. pellucida and Isochrysis galbana, and (iv) N. sp. 2526 whole cell cultures were cultured in glass flasks with 150 mb medium for nine days. Sub-samples of whole-cell culture were removed every few days for bromoform concentration, haloperoxidase activity, and cell concentration. On day 9, the cells were harvested and the cells were separated from the cell-free culture medium by a standard centrifugation method. Cell concentration was determined by flow cytometry on days 1, 2, 3, 4, 7 and 9.

[0463] Haloperoxidase activity was evaluated by aminophenyl fluorescein (APF) assay on days 1, 4, and 7. Bromoform was quantified by gas chromatography / mass spectrometry (GC / MS) on days 1, 4, 7, and 9. FIG. 2 shows typical flow cytometry results for a co-culture of N. pellucida!!. galbana. FIGS. 13A-B, 16A-B, and 17A-B show flow cytometry results for other co-culturing conditions (N. pellucida co-cultured with Isochrysis galbana and Nitzschia 0303 co-cultured with Rhodomonas galbana. FIGS. 3A-B demonstrate that microalgae growth is attenuated in the presence of competitive microalgal species. FIG.3A shows the cell concentration of N. pellucida over the course of 9 days as a monoculture and as a co-culture with N. sp. 2526 and as a co-culture with I. galbana. N. pellucida concentration beginning on day 4 is reduced relative to the N. pellucida monoculture concentration for the remainder of the experiment. This suggests that Nitzschia cf. pellucida is actively investing in production of halogenated compounds, rather than in growth and reproduction when exposed to competitor algae. FIG 3B shows the cell concentration of N. sp. 2526 over the course of 9 days as a monoculture and as a co-culture with N. pellucida. N sp. 2526 cell concentration substantially increases between days 4 and 7 and then stabilizes between days 7 and 9 at about 250 to 300 X103cells / mL as a monoculture; however, N. sp. 2526 tends to zero in the presence of competing N. pellucida. FIG.3B also demonstrates that I. galbana, when grown as a co-culture with N. pellucida does not grow at all, and immediately starts dying, until it reaches, and is able to maintain a cell concentration around 100,000 cells per ml.

[0464] N. pellucida mixed with competing microalgae produced more bromoform than the corresponding TV. pellucida monoculture, and the bromoform was released from the cell, and accumulates in the medium as indicated in the high concentrations of bromoform in the wholecell culture (surrounding medium and cells) as well as the supernatant (cell-free medium, without cells present), compared to the very low concentrations in the cell pellet. FIG. 4A shows the bromoform content per cell of N. pellucida mono- and co-cultures on day 9 as measured in the whole cell culture, supernatant, and cell pellet as quantified by APF assay. Bromoform was predominantly distributed in the whole cell culture and supernatant, whereas only a small fraction was located in the cell pellet. Both the N. pellucida monoculture whole-cell and supernatant samples had approximately 0.2 pg bromoform / cell, whereas the N. pellucida / N. sp.

[0465] 2526 co-culture whole-cell and supernatant samples had approximately 0.5 pg / bromoform / cell and the N. pellucida!!. galbana co-culture whole-cell and supernatant samples had approximately O.35 pg bromoform / cell. FIG. 4B demonstrates that a relatively small amount of bromoform is distributed to the cell pellet, with the N. pellucida monoculture, and N. pellucida / N. sp. 2526 and N. pellucida! I. galbana co-cultures having bromoform concentration of approximately 0.001 pg / cell, 0.005 pg / cell, and 0.0025 pg / cell, respectively. FIG. 12 further shows exemplary results of the amount of bromoform recovered from a method for removing cells from cell culture, centrifuging to obtain a supernatant, and then filtering through a 0.2 pm filter.

[0466] Bromoform concentration continued to increase during the course of the experiment, reaching its maximum level on about day 7. Bromoform content per cell, however, reached a maximum on day 4. Consistent with other findings, both bromoform concentration over time and bromoform per cell were elevated in the N. pellucida / N. sp. 2526 and N. pellucida / I. galbana cocultures relative to the N. pellucida monoculture. FIG. 5A demonstrates that bromoform concentration peaks on day 7 at approximately 8 ng / ml, 13 ng / ml, and 10 ng / ml for the N. pellucida monoculture, and N. pellucida N. sp. 2526 and N. pellucida 1. galbana co-cultures, respectively. Interestingly, bromoform concentration was highest when N. pellucida was cocultured with another Nitzschia species, namely, N. sp. 2526. FIG. 5B shows that bromoform per cell attained a maximum on day 4 and was significantly greater in the co-culture samples, especially the N. pellucida / N. sp. 2526 co-culture sample. The bromoform per cell on day 4 was approximately 0.5 pg / cell, 4.5 pg / cell, and 1.8 pg / cell for the N. pellucida monoculture, and N. pellucida / N. sp. 2526 and N. pellucida / I. galbana co-cultures, respectively. Again, bromoform per cell was highest when the microalgae was co-cultured with another Nitzschia species, N. sp.

[0467] 2526.

[0468] Haloperoxidase activity per cell correlated with bromoform content per cell (normalized to cell concentration at the time of sampling), with enzymatic activity likewise attaining a maximum value on day 4 as measured by APF assay. FIG. 6 shows that haloperoxidase activity was maximized on day 4, with the N. pellucida monoculture and N. pellucida / N. sp. 2526 and N. pellucida / I. galbana co-cultures having haloperoxidase activity of approximately 0.5, 3.5 and 2, respectively, relative to a control haloperoxidase activity. N. sp. 2526 monoculture was also included and, as shown, had little to no measurable haloperoxidase activity relative to control.

[0469] Without being bound by theory, N. pellucida may secrete bromoform as well as a suite of other halogenated compounds as allelochemicals into the surrounding milieu as it competes for growth and biofdm stability in the face of competing species, especially other Nitzschia species such as N. sp. 2526.

[0470] A further aspect of the study was to investigate the ability of ‘competitive’ microalgal strains to ‘capture’ or sequester small, halogenated organic compounds such as bromoform and dibromomethane from the supernatant by employing strains with high lipid content, e.g., Isochrysis galbana. Although I. galbana failed to effectively ‘capture’ bromoform in the cell pellet after centrifugation, elevated amounts of dibromomethane were found together with dead I. galbana cellular material (i.e., as part of the whole cell sample). FIG. 7A shows the dibromomethane (DBM) concentration in the N. pellucida monoculture, and N. pellucida / N. sp.

[0471] 2526 and N. pellucida! 1. galbana co-cultures. FIG. 7B shows the distribution of DBM in the whole cell culture, as well as the supernatant, and cell pellet fractions in the N. pellucida monoculture, and N. pellucida / N. sp. 2526 and N. pellucida / I. galbana co-cultures after 9 days, with significantly higher DBM concentrations in the N. pellucidall. galbana whole cell sample and supernatant fractions.

[0472] Example 2. Evaluation of Bromoform Production Rate and Loss in a Microalgae Culture Medium

[0473] The example set forth below describes methods to evaluate the production rate of small, halogenated organic compounds, e.g., bromoform, and loss in a microalgae culture. Briefly, N. pellucida was cultured as in Example 1. The amount of bromoform was determined as previously described using solvent extraction and GC / MS. The whole-cell culture and supernatant removed by centrifugation corresponding to the same original culture, were separated in triplicate glass bottles were stored in an incubator at 20°C with a light: dark cycle of 14:10. The bromoform concentration was measured approximately 0 h, 2 h, and 20 h after centrifugation. As shown in FIG. 8, there is negligible loss of bromoform after 2 h and only very marginal loss after 20 h in the supernatant. In the whole cell sample, the bromoform concentration increases after 20 h; however, the cell density in the whole cell sample decreases from about 79,000 cells / mL to about 73, 667 cells / mL. The bromoform production rate, assuming approximately 75,000 cells, was therefore calculated to about 0.168 ng / (mL*h), or about 0.00270 pg / cell / h, with an initial rate of loss of about 0.00424 pg / cell / h. As shown in FIG.

[0474] 8, the bromoform loss was assessed in the supernatant and whole cell samples and a bromoform control of pure bromoform in LI culture medium on days 0, 1, 2, 3, 4, and 7 post-centrifugation.

[0475] In addition to determining the bromoform production and loss rates, the stability during storage at -80°C was assessed. This was mainly done to provide confidence in our measurements after storage at -80C when extracting fresh samples was not possible, and storage was necessary. Whole cell cultures were aliquoted into 4.5 ml subsamples and stored at -80C for various amounts of time. Bromoform concentration was measured immediately after centrifugation and 1 week after storing at -80°C. As demonstrated in FIG. 10, there was no significant change in the bromoform concentration after 1-week storage at -80°C. FIG. 11 shows results from an extended stability test in culture medium (supernatant) demonstrating that bromoform produced by N. pellucida is lost at the same rate as pure bromoform in culture medium under culture conditions (20°C, light dark 14h:10h, and 100 pmol photon light levels). Example 3. Bromoform Production in a Bioreactor Apparatus

[0476] The example set forth below describes methods for production of bromoform in a bioreactor apparatus employing inducible allelopathy. The bioreactor apparatus is contemplated to include one or more reactor vessels and optionally other vessels performing various unit operations, such as extraction or recovery. Preliminary experiments establish that a competitor microalga can increase and induce production of bromoform by exposure to the exudate or supernatant of the competitor algae. This may allow for the culturing of the two microalgae species in separate reactor vessels which allow for the passage of cell-free medium between them. Further investigation has shown that removal of bromoform-enriched medium from the bromoform-producing microalgae increases bromoform production rate. Bromoform produced by microalgae can be extracted directly from the culture media into canola oil, (through vortexing and centrifuging). Finally, other experiments show that bromoform-enriched media can be bubbled through oil and the bromoform can be harvested. It is contemplated that the bromoform-enriched media could be removed from the reactor vessel and transported to an extractor for bromoform harvesting.

[0477] Experiments were initially carried out to determine if the cell-free medium (exudate or supernatant) removed from a TV. sp. (CCMP-2526) culture was sufficient to increase bromoform production in a TV. pellucida 0303 culture. FIG. 18A shows that bromoform concentration (ng mT1) increased from days 3 to 12 when N. pellucida 0303 was co-cultured with N. sp.

[0478] (CCMP-2526) or grown in the cell-free sup ematant / exudate from N. sp. (CCMP-2526), suggesting a chemical cue may have been present inducing bromoform production. FIG. 18B illustrates that the N. pellucida 0303 cell concentration ( IO3cells mT1) and bromoform concentration (ng ml-1) increased together, with a subsequent lag in the drop of bromoform after Day 24.

[0479] Removal of the bromoform-containing supernatant from a TV pellucida 0303 culture, and replenishing with bromoform-free medium, showed that this could trigger an increase in bromoform production by N. pellucida. N. pellucida 0303 was grown as a monoculture and increasing percentages of the cell-free medium was removed and replenished with fresh LI medium while the bromoform production rate was monitored. FIG. 19A illustrates that the maximal bromoform production rate (ng ml’^d’1) in the co-culture was accomplished when approximately 60% of the cell-free medium is removed. FIG. 19B depicts the results of a subsequent experiment in which bromoform production rate (ng ml^ d’1) in a N. pellucida 0303 continued to increase up to 80% cell-free medium removed after supernatant removal. The supernatant was removed once a day for six days.

[0480] Next, a lab scale extractor, comprising 220 mL canola oil in a graduated cylinder and 700 mb aqueous phase containing bromoform in an Erlenmeyer flask, was devised for harvesting bromoform, as shown on FIG. 21A. The graduated cylinder and Erlenmeyer flask were connected by polypropylene tubing and an air stone was used to bubble the aqueous phase, purging the bromoform out of the aqueous phase and thereby allowing the bromoform to accumulate in the canola oil. FIG. 21B demonstrates that the bromoform concentration increased from near 0 to above 2000 ng / mL after approximately 4.5 h, in the oil phase, whereas the bromoform concentration in the aqueous phase drops from about 5500 ng / mL to about 500 mL. Further scaled developments of the extractor lengthen the contact time of halogen-laden gas bubbles with the oily or aqueous solution, thereby increasing transfer and purification rates as show in FIG. 22.

[0481] The bioreactor apparatus (FIG.20) will comprise two reactors, an extractor, and a recovery vessel. The first reactor vessel will provide conditions for the growth of the competitor microalgae Nitzschia sp. (CCMP-2526) and the accumulation of the competitor cue for modulating bromoform production. The first reactor will be fluidly connected to the second reactor, transporting the cell-free medium with the cue. The second reactor vessel will provide for growth of N. pellucida 0303 and accumulation of bromoform. The second reactor vessel will be fluidly connected to the separator, transporting cell-free, bromoform-rich medium to the extractor. The extractor will extract bromoform from the cell-free medium by means of dripping the bromoform-rich medium through a carrier oil sitting atop an aqueous phase. The carrier oil will be bubbled through the aqueous phase from the bottom. Spent medium will be sent to the recovery vessel, where it will be enriched with nutrients, pH rebalanced, stripped of volatiles, and mixed with carbon dioxide-enriched air. The volatiles will be sent to a scrubber and the recovered medium will be recycled to the first reactor.

[0482] In another aspect, it is contemplated that the apparatus comprises an extractor for purging the bromoform using air and bubbling it through the oil, rather sending the liquid through the oil. EQUIVALENTS

[0483] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific aspects, it is apparent that other aspects and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.

Claims

CLAIMS1. A method for producing a small, halogenated organic compound from microalgae via a halogenation reaction, the method comprising:(i) preparing a culture of microalgae; and(ii) acquiring information about the small, halogenated organic compound,thereby producing a small, halogenated organic compound.

2. A method for producing a small, halogenated organic compound from microalgae via a halogenation reaction, the method comprising:(i) preparing a culture of microalgae;(ii) optionally contacting the culture with a halogen source and a small organic compound to form a mixture;(iii) optionally mixing the mixture; and(iv) acquiring information about the small, halogenated organic compound,thereby producing a small, halogenated organic compound.

3. The method of claim 1, wherein the culture is a monoculture.

4. The method of claim 1, wherein the culture is a co-culture, e.g., the culture comprises a plurality of microalgae strains.

5. A method for modulating the production of a small, halogenated organic compound via a halogenation reaction, the method comprising:(i) preparing a co-culture of microalgae, thereby initiating a halogenation reaction;(ii) acquiring information about the small, halogenated organic compound; and(iii) extracting the small, halogenated organic compound from the co-culture, thereby producing a small, halogenated organic compound.

6. The method of claim 1, wherein the microalgae comprises a cyanobacterium, a diatom, a haptophyte, chlororachniophyte, ulvophyte or a dinoflagellate.

7. The method of claim 6, wherein the microalgae are selected from Nitzschia cf pellucida, Isochrysis galbana Porosira glacialis, Bigelowiella longifila, Ulvella scutata, Ditylum brightwellii, Nitzschia laevis, or a combination thereof.

8. The method of claim 1, wherein the microalgae are capable of forming a biofilm.

9. The method of claim 1, wherein the microalgae comprise a high lipid content, e.g., a lipid content of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., 10-40% lipid content).

10. The method of claim 1, wherein the microalgae comprising a high lipid content is selected from Botryococcus braunii, Isochrysis galbana, Neochloris oleoabundans, Phaeodactylum tricornutum, Pleurochrysis carterae, Prymnesium parvum, Tetradesmus dimorphus, Tetraselmis chui, Tetraselmis \necica. and Tisochrysis lutea, or a combination thereof.

11. The method of claim 1, wherein the small halogenated organic compound has a structure of Formula (Z):or a salt, tautomer, or isomerR5a, R5b, and R5cis independently hydrogen, halogen, C1-C6alkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, or heterocyclyl, wherein each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl is optionally substituted with one or more R6, and at least one of Rla, Rlb, Rlc, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, and R5cis independently halogen; R6is halogen, C1-C6alkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, -ORA, or -NRBRC; RAis hydrogen, C1-C6alkyl, C1-C6heteroalkyl, or C2-C6alkenyl; RBand RCare each independently hydrogen, C1-C6alkyl, or C1-C6heteroalkyl; each of m and n is independently selected from 0, 1,2, or 3; and “ - ” is a single or double bond, wherein when - is a double bond, each of R2band R3bis independently absent.

12. The method of claim 1, wherein the small halogenated organic compound is chlorinated; brominated; iodinated; chlorinated and iodinated; chlorinated and brominated; brominated and iodinated; or chlorinated, brominated, or iodinated.

13. The method of claim 12, wherein the small halogenated organic compound is brominated.

14. The method of claim 1, wherein the small halogenated organic compound comprises 1, 2, or 3 halogen atoms.

15. The method of claim 14, wherein the small halogenated organic compound comprises 1, 2, or 3 bromine atoms.

16. The method of claim 1, wherein the small halogenated organic compound comprises an acetone moiety.

17. The method of claim 1, wherein the small halogenated organic compound comprises dibromomethane, dibromoacetone, bromopentanedione, bromoform, or tribromoacetone.

18. The method of claim 1, wherein the small halogenated organic compound comprises di chloroiodomethane, dibromochloromethane, 1,1 -dibromoacetone, bromoacetone, dibromomethane, 3-bromo-2,4-pentanedione, bromoform, 1,3 -tribromoacetone, or 1,1,1-tribromoacetone.

19. The method of claim 1, wherein the microalgae is allelopathic or is capable of producing an allelochemical.

20. The method of claim 1, wherein the allelochemical is a small halogenated organic compound.

21. The method of claim 1, the method further comprising sequestering the small halogenated organic compound with a microalgae strain.

22. The method of claim 21, wherein the sequestering comprises culturing a microalgae strain with a high lipid content, e.g., a lipid content of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (e.g., 10-40% lipid content).

23. The method of claim 22, wherein the microalgae strain with a high lipid content is Isochrysis galbana.

24. The method of any one of the preceding claims, wherein the acquiring comprises:(i) characterizing the small halogenated organic compound, e.g., by gas chromatography or mass spectrometry;(ii) characterizing the microalgae, e.g., the cell concentration, e.g., by flow cytometry, or cell viability, e.g., by oxygen evolution and primary productivity rates, LIVE / DEAD assay; and / or(iii) characterizing the haloperoxidase activity, e.g., by aminophenyl fluorescein (APF) assay.

25. The method of claim 1, wherein the method further comprises (v) separating the small halogenated organic compound from the mixture.

26. The method of claim 25, wherein the (v) separating comprises centrifuging the mixture, e.g., centrifuging the mixture into cell pellet and supernatant fractions.

27. The method of claim 25, wherein the (v) separating comprises extracting the small, halogenated organic compound, e.g., by solvent extraction.

28. The method of claim 25, wherein the (v) separating comprises extracting with an oil, e.g., canola oil.

29. The method of claim 1, wherein the cell density of the culture is between about 102to 106cells / mL, e.g., about 102, 103, 104, 105, 106, 107, 108, or 109cell / mL.

30. The method of claim 1, wherein the temperature of the culture is between about 10°C to 50°C, e.g., 20°C, 25°C or37°C.

31. The method of claim 1, wherein the pH of the culture is between about 4 to 10, e.g., about 4, 5, 6, 7, 8, 9, or 10.

32. A method for reducing production of methane in a rumen community, the method comprising:(i) preparing a culture of microalgae;(ii) contacting the culture with a halogen source and a small organic compound to form a mixture;(iii) mixing the mixture; and(iv) separating the small halogenated organic compound from the mixture; and(v) providing the small halogenated organic compound to a rumen community under conditions sufficient to reduce the production of methane.

33. The method of claim 32, further comprising acquiring a value for the level of methane prior to the contacting of step (ii).

34. The method of claim 32, wherein the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.

35. The method of claim 34, wherein the methane production is reduced by between 10-75%.

36. A method for reducing production of methane in a manure management system, the method comprising:(i) preparing a culture of microalgae;(ii) contacting the culture with a halogen source and a small organic compound to form a mixture;(iii) mixing the mixture;(iv) separating the small halogenated organic compound from the mixture;(v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane.

37. The method of claim 36, further comprising acquiring a value for the level of methane prior to the contacting of step (ii).

38. The method of claim 36, wherein the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.

39. A method for preparing animal feed for reducing the production of methane in a rumen community, the method comprising:(i) preparing a culture of microalgae;(ii) contacting the culture with a halogen source and a small organic compound to form a mixture;(iii) mixing the mixture;(iv) separating the small halogenated organic compound from the mixture; and(v) preparing a composition comprising a small halogenated organic compound suitable for use as an animal feed,thereby preparing animal feed for reducing the production of methane in a rumen community.

40. A method for reducing production of methane in a rumen community, the method comprising:(i) preparing a culture of microalgae;(iv) separating the small halogenated organic compound from the mixture; and(v) providing the small halogenated organic compound to a rumen community under conditions sufficient to reduce the production of methane.

41. The method of claim 40, wherein the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.

42. The method of claim 40, wherein the methane production is reduced by between 10-75%.

43. A method for reducing production of methane in a manure management system, the method comprising:(i) preparing a culture of microalgae;(iv) separating the small halogenated organic compound from the mixture; and(v) providing the small halogenated organic compound to a manure management system under conditions sufficient to reduce the production of methane.

44. The method of claim 43, wherein the methane production is reduced by at least 5%, 10% 15%, 20%, 25% 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more.

45. A method for preparing animal feed for reducing the production of methane in a rumen community, the method comprising:(i) preparing a culture of microalgae;(iv) separating the small halogenated organic compound from the mixture; and(v) preparing a composition comprising a small halogenated organic compound suitable for use as an animal feed,thereby preparing animal feed for reducing the production of methane in a rumen community.

46. An apparatus for culturing a microalgal species, comprising a vessel for producing a small, halogenated organic compound.

47. The apparatus of claim 46, wherein the vessel is a reactor vessel, a separator vessel (e.g., an extractor), or a recovery vessel.

48. The apparatus of claim 46, wherein the extractor comprises an aqueous phase and an oily phase.

49. The apparatus of claim 46, wherein the extractor comprises a gaseous phase and an aqueous phase.

50. The apparatus of claim 46, comprising a plurality of vessels, e.g., reactor vessels.

51. The apparatus of claim 47, wherein each vessel in the plurality is fluidly connected to at least one of the vessels in the plurality.

52. The apparatus of claim 46, wherein the first reactor vessel comprises microalgae exuding a particular cue, and the second reactor vessel comprises microalgae capable of modulating production of a small, halogenated organic compound responsive to the competitor cue.

53. An apparatus for producing a small, halogenated organic compound: the apparatus comprising a plurality of vessels:a first reactor vessel comprising a first microalga capable of exuding an allelochemical allelochemical cue, that can be detected by competitor algae;a second reactor vessel comprising a second microalgae capable of modulating production of a small, halogenated organic compound responsive to the competitor cue;an extractor for harvesting the small, halogenated organic compound; anda recovery vessel for nutrient enrichment, pH correction, or volatile compound stripping; and, wherein each vessel is fluidly connected to at least one of the vessels in the plurality.

54. The apparatus of claim 53, wherein the small, halogenated organic compound is bromoform.

55. The apparatus of claim 46, wherein the reactor vessel is a batch reactor, a semi-batch reactor, or a continuous reactor, e.g., a continuous stirred-tank reactor (CSTR).

56. The apparatus of claim 55, wherein the reactor vessel is the batch reactor.

57. The apparatus of claim 55, wherein the reactor vessel is the semi-batch reactor.

58. The apparatus of claim 55, wherein the reactor vessel is the continuous reactor, e.g., the continuous stirred-tank reactor (CSTR).

59. The apparatus of claim 46, wherein the reactor vessel is operated at steady state or unsteady state.

60. The apparatus of claim 59, wherein the reactor vessel is operated at steady state.

61. The apparatus of claim 59, wherein the reactor vessel is operated at unsteady state.

62. The apparatus of claim 46, wherein the reactor vessel comprises an impeller or a stirrer.

63. The apparatus of claim 62, wherein the impeller comprises one or more baffles.

64. The apparatus of claim 62, wherein the impeller or stirrer provides for laminar flow conditions, e.g., suitable for growth of the microalgae and / or removal or addition of media, e.g., media comprising a chemical cue (e.g., allelochemical), e.g., a small halogenated organic compound, e.g., bromoform.

65. The apparatus of claim 46, wherein the reactor vessel is a metal (e.g., metal alloy, e.g., steel), glass (e.g., borosilicate), plastic, or ceramic vessel.

66. The apparatus of claim 46, wherein the reactor vessel is cylindrical, rectangular, spherical, ellipsoidal, toroidal, prismatic, or pyramidal.

67. The apparatus of claim 46, wherein the reactor vessel is transparent or translucent.

68. The apparatus of claim 46, wherein the reactor vessel comprises an inlet or an outlet, e.g., for addition or removal of a liquid (e g., cell media), gas (e.g., CCh-enriched air), or solid (e.g., cells).

69. The apparatus of claim 46, wherein the reactor vessel is vented (e g., from the top, side, or bottom of the vessel, e.g., with the outside atmosphere).

70. The apparatus of claim 46, wherein the reactor vessel is hermetically sealed.

71. The apparatus of claim 46, wherein the reactor vessel further comprises an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) for dispersing small particles, e.g., liquid droplets (e.g., of oil or water) or bubbles (e.g., air bubbles compromising a small, halogenated organic compound, e.g., bromoform).

72. The apparatus of claim 51, wherein the reactor vessels are fluidly connected with tubing, thereby allowing transporting of a medium (e.g., a liquid, solid, gas, or a mixture thereof) between the two or more vessels.

73. The apparatus of claim 72, wherein the tubing is hermetically sealed.

74. The apparatus of claim 72, wherein the tubing is comprised of a metal, or plastic (e.g., polypropylene).

75. The apparatus of claim 72, wherein the medium comprises a supernatant, e.g., cell-free medium, e.g., cell-free medium comprising a small, halogenated organic compound, e.g., bromoform.

76. The apparatus of claim 72, wherein the medium is an aqueous medium enriched with nutrients, stripped of volatiles, and mixed with CO₂-enriched air suitable for culturing of microalgae.

77. The apparatus of claim 72, wherein the medium does not comprise cells.

78. The apparatus of claim 72, wherein the medium is aqueous.

79. The apparatus of claim 72, wherein the medium comprises an oil.

80. The apparatus of claim 72, wherein the medium comprises an emulsion of two or more aqueous and oil phases.

81. The apparatus of claim 72, wherein the medium comprises bubbles, e.g., air bubbles, e.g., air bubbles comprising volatiles, e.g., small halogenated organic compounds, e.g., bromoform.

82. The apparatus of claim 46, wherein the separator vessel comprises an extractor vessel.

83. The apparatus of claim 82, wherein the extractor vessel comprises two or more aqueous and oil phases.

84. The apparatus of claim 82, wherein the extractor vessel is operated at steady state or unsteady state.

85. The apparatus of claim 82, wherein the extractor vessel is operated at steady state.

86. The apparatus of claim 82, wherein the extractor vessel is operated at unsteady state.

87. The apparatus of claim 82, wherein the extractor vessel comprises an impeller or a stirrer.

88. The apparatus of claim 82, wherein the impeller comprises one or more baffles.

89. The apparatus of claim 82, wherein the impeller or stirrer provides for laminar flow conditions, e.g., suitable for growth of the microalgae and / or removal or addition of media, e.g., media comprising a chemical cue (e.g., allelochemical), e.g., a small halogenated organic compound, e.g., bromoform.

90. The apparatus of claim 82, wherein the extractor vessel is a metal (e.g., metal alloy, e.g., steel), glass (e.g., borosilicate), plastic, or ceramic vessel.

91. The apparatus of claim 82, wherein the extractor vessel is cylindrical, rectangular, spherical, ellipsoidal, toroidal, prismatic, or pyramidal.

92. The apparatus of claim 82, wherein the extractor vessel is transparent or translucent.

93. The apparatus of claim 82, wherein the extractor vessel comprises an inlet or an outlet, e.g., for addition or removal of a liquid (e.g., cell media), gas (e.g., CO₂-enriched air), or solid (e.g., cells).

94. The apparatus of claim 82, wherein the extractor vessel is vented (e.g., from the top, side, or bottom of the vessel, e.g., with the outside atmosphere).

95. The apparatus of claim 82, wherein the extractor vessel is hermetically sealed.

96. The apparatus of claim 82, wherein the extractor vessel further comprises an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) for dispersing small particles, e.g., liquid droplets (e.g., of oil or water) or bubbles (e.g., air bubbles compromising a small, halogenated organic compound, e.g., bromoform).

97. The apparatus of claim 82, wherein the extractor vessel is fluidly connected to one or more vessels in the apparatus with tubing, thereby allowing transporting of a medium (e.g., a liquid, solid, gas, or a mixture thereof) between the two or more vessels.

98. The apparatus of claim 97, wherein the tubing is hermetically sealed.

99. The apparatus of claim 97, wherein the tubing is comprised of a metal, or plastic (e g., polypropylene).

100. The apparatus of claim 97, wherein the medium comprises a supernatant, e.g., cell-free medium, e.g., cell-free medium comprising a small, halogenated organic compound, e.g., bromoform.

101. The apparatus of claim 97, wherein the medium is an aqueous medium enriched with nutrients, stripped of volatiles, and mixed with CO₂-enriched air suitable for culturing of microalgae.

102. The apparatus of claim 97, wherein the medium does not comprise cells.

103. The apparatus of claim 97, wherein the medium is aqueous.

104. The apparatus of claim 97, wherein the medium comprises an oil.

105. The apparatus of claim 97, wherein the medium comprises an emulsion of two or more aqueous and oil phases.

107. The apparatus of claim 97, wherein the medium comprises bubbles, e.g., air bubbles, e.g., air bubbles comprising volatiles, e.g., small halogenated organic compounds, e.g., bromoform.

108. The apparatus of claim 82, wherein the extractor vessel comprises an aqueous phase fluidly connected with the oil phase.

109. The apparatus of claim 108, wherein the aqueous phase and the oily phase are in the same vessel or separate vessels.

110. The apparatus of claim 109, wherein the aqueous phase is enriched with the small, halogenated organic compound (e.g., bromoform).

111. The apparatus of claim 109, wherein the partition coefficient in octanol / water of the small, halogenated organic compound (e.g., bromoform) favors accumulation of the small, halogenated organic compound (e.g., bromoform) in the oil phase at equilibrium.

112. The apparatus of claim 109, wherein the oil comprises canola (rapeseed) oil, olive oil, palm oil, sunflower oil, or avocado oil.

113. The apparatus of claim 109, wherein the aqueous phase contains an atomizer, nebulizer, aerosolizer, or bubbler (e.g., an airstone) to induce air bubble formation.

114. The apparatus of claim 46, comprising a recovery vessel.

115. The apparatus of claim 114, wherein the recovery vessel provides for nutrient enrichment, pH correction or rebuffering, or volatile compound stripping.

116. The apparatus of claim 115, wherein the recovery vessel provides for nutrient enrichment.

117. The apparatus of claim 115, wherein the recovery vessel provides for pH correction or rebuffering.

118. The apparatus of claim 115, wherein the recovery vessel provides for volatile compound stripping.

119. The apparatus of claim 46, wherein the small, halogenated compound, e.g., bromoform, is purged out of a reactor vessel for harvesting into an organic phase, e.g., oil phase, e.g., by liquid gas extraction.

120. The apparatus of claim 119, wherein an extractor vessel performs liquid-gas extraction, thereby harvesting the small, halogenated organic compound.

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