Field of the Invention

The present invention relates to surface active agents, in particular surface active agents obtainable from natural sources, methods of producing said surface active agents and the use of said surface active agents.

Background

Surface-active agents, for example, emulsifiers and surfactants, constitute an important class of chemicals used in almost every sector of modern industry. At present, market demand for surfactants is met almost exclusively by synthetic, petroleum- based compounds. These petroleum-based compounds suffer from the disadvantage that, in the future, petroleum supplies may diminish. Further, many of the existing surface active agents have been determined to be environmentally problematic e.g. due to poor biodegradability.

In view of the environmental problems associated with synthetically produced surface active agents, surface active agents with emulsifying and / or surfactant activity which are derivable from natural and sustainable sources have been sought e.g. Xanthan gum. Typically such surface active agents are referred to as bio-emulsifiers and / or bio- surfactants.

Bioprospecting for natural products which show advantageous characteristics is well known, but the labour intensive nature of bioprospecting and the vast number of potential targets creates difficulties in isolating, obtaining and / or identifying bacteria and derived bacterial products from natural sources. A need exists for further surface active agents.

Summary of the invention

The inventors of the present invention have identified phycospheric bacteria that surprisingly are able to produce surface active agents which act as bio-surfactants and / or bio-emulsifiers and, moreover, some of which have comparable if not advantageous activity when compared to various commercially used emulsifiers such as Xanthan Gum.

Accordingly, a first aspect of the present invention provides a method of reducing surface tension in a liquid and / or producing and stabilising a liquid emulsion, said method comprising adding to said liquid an effective amount of a surface active agent or a synthetic or recombinant equivalent thereof, said agent being obtainable as extract from phycospheric bacteria.

Suitably the surface active agent is obtained as an extract of phycospheric bacteria.

For the purposes of the present invention, phycosphere is a zone around freshwater and / or marine macro or micro algae in which bacteria are influenced by algae and conversely algae by bacteria.

For the purposes of the present invention, phycospheric bacteria are bacteria which, when in their natural aquatic environment, inhabit the phycosphere.

Bacteria can be free in the phycosphere, attached to the surface of algal cells or intracellular to the algae.

In particular embodiments the bacteria are obtainable from the surface of algae which may be macro-algae or micro-algae. In specific embodiments the bacteria are obtainable from the surface of micro-algae.

Micro-algae are unicellular photosynthetic plants.

Macro-algae are multicellular eukaryotic algae.

In embodiments of the invention the bacteria are obtainable from marine algae.

In specific embodiments at least one of the bacteria of or for use in the invention is of the genus Pseudoalteromonas, Antarctobacter or Halomonas.

In specific embodiments the bacteria is of a similar strain as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41242 (hereinafter referred to as SAMS 12), as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41245 (hereinafter referred to as SAMS 22), as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41244 (hereinafter referred to as SAMS 39), or as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41243 (hereinafter referred to as SAMS 67) or is a surface active agent-producing recombinant bacteria, mutant or variant of any one of these strains or any B2005/00342!

combination thereof.

By "similar strains of bacteria" we refer to those strains which share greater than 98% genetic homology to each other, more preferably greater than 99% genetic homology to each other.

In preferred embodiments the bacteria is the same strain as the strain as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41242 (SAMS 12) , as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41245 (SAMS 22) , as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41244 (SAMS 39) , or as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41243 (SAMS 67) .

Isolates of strains of the invention were deposited on 1 September 2004 at the National Collection of Industrial and Marine Bacteria, in accordance with the terms of the Budapest Treaty as outlined above.

Optionally the surface active agent may be obtained synthetically or by recombinant means.

In an embodiment of the invention wherein the surface active agent is provided by recombinant means, the gene(s) which provide surface active agents of the invention are determined and suitably provided in a recombinant vector or cell free system which allows the protein(s) of said gene(s) to be expressed and surface active agents to be provided.

In an embodiment wherein a recombinant method is used to produce recombinant surface active agents of the invention, the gene(s) provided in the recombinant vector or cell free system preferably have a sequence of at least 85% homology to the gene(s) determined from the phy.cospheric bacteria of the invention, more preferably at least 90% homology to the gene(s) determined from the phycospheric bacteria of the invention, and yet more preferably at least 95% homology to the gene(s) determined from the phycospheric bacteria of the invention.

In one preferred embodiment, the gene(s) provided in the recombinant vector or cell free system have 100% homology to the genes determined from the phycospheric bacteria of the invention.

In one embodiment, an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of amino acids in the range

List a

Aspartic acid / Asparagine 0.5 to 1.5 Glutamic acid / Glutamine 0.5 to 1.5 Serine 0.1 to 1.0 Glycine 0.1 to 1.0 Histidine 0.05 to 0.5 Threonine 0.05 to 1.0 Arginine 0.05 to 1.0 Alanine 0.05 to 1.0 Proline 0.05 to 0.5 Tyrosine 0.05 to 0.5 Cysteine 0.05 to 1.0 Valine 0.05 to 1.0 Methionine 0.05 to 1.0 Isoleucine 0.05 to 1.0 Leucine 0.05 to 1.0 Lysine 0.05 to 1.0, and Phe 0.05 to 1.0

In one embodiment an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of sugar components in the range

List b

Rha (rhamnose) 0.05 to 2.0 Fuc (fucose) 0.05 to 1.0 Gal (galactose) 0 . 05 to 2 . 0 GaIN ( galactosartiine ) and ^acetylgalactosamine 0 . 05 to 2 . 0 GIc (glucose ) 8 . 0 to 13 . 0 GIcN (glucosamine) and N-acetylglucosamine 20.0 to 30.0 Man (mannose) 2.5 to 6.5 XyI (xylose) 20.0 to 35.0 Mur (muramic acid) 0.05 to 1.0 GaIA (galacturonic acid) 20.0 to 30.0, and GIcA (glucuronic acid) 3.0 to 7.0

Optionally an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of amino acids as detailed in list a and a percent mean composition of sugar components as detailed in list b.

In one embodiment, an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of amino acids in the range

List c

Aspartic acid / Asparagine 0.05 to 1.0 Glutamic acid / Glutamine 0.05 to 1.0 Serine 0.05 to 1.0 Glycine 0.05 to 1.0 Histidine 0.01 to 0.5 Threonine 0.05 to 1.0 Arginine 0.05 to 1.0 Alanine 0.05 to 1.0 Proline 0.05 to 1.0 Tyrosine 0.05 to 1.0 Cysteine 0.01 to 0.5 Valine 0.05 to 1.0 Isoleucine 0.05 to 1.0 Leucine 0.05 to 1.0 Lysine 0.05 to 1.0, and Phe 0.05 to 1.0

In one embodiment, an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of sugar components in the range

List d

Rha (rhamnose) 3.0 to 7.0 Fuc (fucose) 14.0 to 18.0 Gal (galactose) 0.05 to 1.0 GaIN (galactosamine) and N-acetylgalactosamine 0.5 to 2.5 GIc (glucose) 1.5 to 4.0 GIcN (glucosamine) and N-acetylglucosamine 25.0 to 35.0 Man (mannose) 6.55 to 12.5 Mur (muramic acid) 5.5 to 9.5 GaIA (galacturoniσ acid) 2.0 to 6.0, and GIcA (glucuronic acid) 15.0 to 25.0

Optionally an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of amino acids as detailed in list c and a percent mean composition of sugar components as detailed in list d.

In one embodiment, an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of amino acids in the range List e

Aspartic acid / Asparagine 1.5 to 5.0 Glutamic acid / Glutamine 1.5 to 5.0 Serine 0.5 to 2.0 Glycine 0.5 to 2.5 Histidine 0.05 to 1.0 Threonine 0.05 to 3.0 Arginine 0.05 to 3.0 Alanine 0.05 to 3.5 Proline 0.05 to 2.0 Tyrosine 0.05 to 2.0 Cysteine 0.01 to 1.0 Valine 0.5 to 4.0 Methionine 0.05 to 1.0 Isoleucine 0.05 to 4.0 Leucine 0.05 to 4.0 Lysine 0.05 to 4.0, and Phe 0.05 to 4.0

In one embodiment, an extract obtained :from phycospheric bacteria or by recombinant or synth methods comprises a percent mean composition of sugar components in the range

List f

Rha (rhamnose) 25.0 to 35.0 Gal (galactose) 10.0 to 20.0 GaIN (galactosamine) and N-acetylgalactosamine 0.5 to 4.0 GIc (glucose) 5 . 0 to 10 . 0 GIcN (glucosamine) and N-acetylglucosamine 5.0 to 10.0 Man (mannose) 1.5 to 5.0 XyI (xylose) 0.05 to 1.5 GaIA (galacturonic acid) 0.5 to 5.0, and GIcA (glucuronic acid) 22.0 to 33.0

Optionally an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of amino acids as detailed in list e and a percent mean composition of sugar components as detailed in list f.

In one embodiment, an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of amino acids in the range

List g

Aspartic acid / Asparagine 5.0 to 10.0 Glutamic acid / Glutamine 3.0 to 8.0 Serine 0.5 to 5.0 Glycine 0.5 to 5.0 Histidine 0.05 to 1.0 Threonine 1.0 to 5.0 Arginine 0.5 to 4.0 Alanine 1.0 to 5.0 Proline 0.05 to 2.5 Tyrosine 0.05 to 2.5 Cysteine 0.01 to 1.0 Valine 0 . 5 to 5 . 0 Methionine 0.05 to 1.0 Isoleucine 0.05 to 2.5 Leucine 1.0 to 5.0 Lysine 1.05 to 2.5, and Phe 0.5 to 5.0

In one embodiment, an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of sugar components in the range

List h

Rha (rhamnose) 1.0 to 6.0 Gal (galactose) 0.05 to 3.5 GaIN (galactosamine) and N-acetylgalactosamine 2.0 to 7.0 GIc (glucose) 4.0 to 10.0 GIcN (glucosamine) and N-acetylglucosamine 7.0 to 15.0 Man (mannose) 7.5 to 15.0 Mur (muramic acid) 0.01 to 1.0 GaIA (galacturonic acid) 0.5 to 4.0, and GIcA (glucuronic acid) 45.0 to 65.0

Optionally an extract obtained from phycospheric bacteria or by recombinant or synthetic methods comprises a percent mean composition of amino acids as detailed in list g and a percent mean composition of sugar components as detailed in list h. As part of the study in which extracts of phycospheric bacteria have been shown to reduce surface tension in a liquid and / or to stabilise a liquid emulsion, the bacteria have been isolated from their natural surroundings.

Accordingly, a second aspect of the invention provides an isolated novel bacteria of the strain as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41242 (SAMS 12) or a surface active agent-producing recombinant bacteria, mutant or variant thereof, as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41245 (SAMS 22) or a surface active agent-producing recombinant bacteria, mutant or variant thereof, as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41244 (SAMS 39) or a surface active agent-producing recombinant bacteria, mutant or variant thereof, or as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41243 (SAMS 67) or a surface active agent-producing recombinant bacteria, mutant or variant thereof, or any combination of these strains or mutants or variants.

In specific embodiments there is provided a bacteria of the strain as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41242 (SAMS 12), a strain as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41245 (SAMS 22) , a strain as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41244 (SAMS 39) , or as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41243 (SAMS 67), or any combination thereof.

Preferably the bacteria of and for use in the invention are bacteria which are in their natural state, i.e not genetically modified, for example the bacteria may be native bacteria as found in the phycosphere of algae.

The isolated bacteria can be utilised to provide a surface active agent, for example, suitable for use in a method of the first aspect of the invention.

Accordingly a third aspect of the present invention provides a process for the preparation of a surface active agent, said process comprising the step of culturing a phycospheric bacteria and isolating and, optionally, purifying a surface active agent from said bacteria.

Preferably said surface active agent is produced by native bacteria which in their natural environment naturally produce surface active agent. The process for the preparation of a surface active agent may be enhanced by modification of at least one parameter selected from the group consisting of temperature, pressure, dissolved oxygen concentration, pH adjustment, growth media and / or combinations of said parameters.

The skilled person would understand how such parameters may be manipulated to increase the yield and / or improve the rate of production of a surface active agent.

The invention further extends to a fourth aspect of the invention wherein there is provided novel surface active agents obtainable from phycospheric bacteria.

In one embodiment, said surface active agent is obtainable from a phycospheric bacteria of genus Pseudoalteromonas, Antarctobacter or Halomonas.

In a preferred embodiment said surface active agent (s) is obtainable from bacteria of a strain as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41242 (SAMS 12), as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41245 (SAMS 22), as deposited the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41244 5 003421

16

(SAMS 39), or as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41243 (SAMS 67) or a surface active agent-producing recombinant bacteria, mutant or variant thereof of any one of these strains.

Surface active agents of the invention may be produced using the method of the third aspect of the invention or may be chemically synthesised.

In one embodiment the surface active agent is obtained from bacteria. Suitably the surface active agent may be obtained from bacteria of a strain as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41242 (SAMS 12), as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41245 (SAMS 22), as deposited the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41244 (SAMS 39), or as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41243 (SAMS 67) or a surface active agent-producing recombinant bacteria, mutant or variant thereof of any one of these strains.

In one embodiment a surface active agent comprising a mean composition of amino acids as detailed in list a, list c, list e or list g is provided. In one embodiment a surface active agent comprising a mean composition of sugar components as detailed in list b, list d,- list f or list h is provided.

In one embodiment a surface active agent comprising a mean composition of amino acids as detailed in list a and a mean composition of sugar components as detailed in list b is provided.

In one embodiment a surface active agent comprising a mean composition of amino acids as detailed in list c and a mean composition of sugar components as detailed in list d is provided.

In one embodiment a surface active agent comprising a mean composition of amino acids as detailed in list e and a mean composition of sugar components as detailed in list f is provided.

In one embodiment a surface active agent comprising a mean composition of amino acids as detailed in list g and a mean composition of sugar components as detailed in list h is provided.

In one embodiment determination of structural characteristics of the surface active agent produced by the bacteria of the invention may allow the surface active agent or analog(s) thereof to be partially or entirely chemically synthesised. The surface active agent of the invention may be further modified to improve the stability or activity of the surface active agent.

Further, in another embodiment the surface active agent may be obtained in precursor form prior to being modified, e.g. synthetically modified to provide a surface active agent with desired characteristics.

According to a fifth aspect of the present invention there is provided the use of a phycospheric bacteria as a source of surface active agent.

According to a sixth aspect of the present invention there is provided the use of an agent obtainable from a cell extract of phycospheric bacteria as a surface active agent.

In embodiments of the invention the cell extract may include the whole bacterial cell.

In alternative embodiments the cell extract includes extracellular agents or agents secreted by the cell.

According to a seventh aspect of the invention there is provided an oil recovery composition, said composition comprising a surface active agent of the invention.

In one embodiment the extract may be the bacteria as a whole. The bacteria, and/or the surface active 2005/00342!

19

agents could be injected in the form of a liquid, or in the form of a dried sample, to underground wells to enhance oil recovery.

According to an eighth aspect of the invention there is provided a composition for coating surfaces to minimise the attachment and / or growth of organisms, said composition comprising a surface active agent according to a fourth aspect of the invention.

According to an ninth aspect of the invention there is provided a bioremediation composition, said composition comprising a surface active agent of the invention.

The bioremediation composition may be utilised to clean up contaminants from contaminated sites, for example in separation and recovery of hydrocarbons and / or hydrocarbon and heavy metal removal.

According to a tenth aspect of the invention there is provided a live seed culture, said culture comprising an isolated bacteria of the strain as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41242 (SAMS 12), as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41245 (SAMS 22), as deposited at the National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41244 (SAMS 39) , or as deposited at National Collection of Industrial and Marine Bacteria NCIMB on 1 September 2004 under Accession number 41242 (SAMS 67), or a surface active agent-producing recombinant bacteria, mutant or variant thereof of any one of these isolates and combinations thereof.

Live seed cultures of bacteria of the present invention may be provided to contaminated sites such that the bacteria enhance the clean up of contaminants from the contaminated site.

Bacteria for use in the present invention may be isolated from algae using any suitable method.

According to an eleventh aspect of the present invention there is provided a surface active agent of the present invention in a form suitable for use in food processing, for example in washing equipment for food preparation or foods (in particular fruit and vegetables) and / or to solubilise flavouring oils introduced into food.

According to a twelfth aspect of the present invention there is provided a surface active agent of the present invention in a form suitable for use in cleaning products, for example within washing powders or detergent formulations and products suitable for cleaning solid and soft surfaces.

According to a thirteenth aspect of the present invention there is provided a surface active agent of the present invention in a form suitable for use in healthcare products, cosmetics and pre-biotic formulations.

As well as the applications described above, the surface active agent of the invention may find use in other purposes for which conventional surface active agents are used. For example, the surface active agents may be used, but not limited to, in agriculture for use as a wetter and / or adjuvant or as a fungicide, in construction for use in surfacing or in concrete, in cosmetics or personal care for use in formulations i.e. lipstick and shampoo, in polymers and plastics, in electronic circuit boards, to enhance foaming in fire control products, in cleaning products, in inks, in leather production, in metal production, (ore production, casting, coating and plating) , in paints and protective coatings such as waxes and polishes, in preparation and / or the dying of textiles and in water treatment.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis unless the context demands otherwise.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention. Throughout the specification, unless the context demands otherwise, the terms ^comprise' or ^include', or variations such as ^comprises' or ^comprising' , λincludes' or ^including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

Brief description of the drawings

The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention, and further, with reference to the figures.

Figure 1 is a graph showing the production of emulsifier by isolate 12 in different media and that glucose is required for the production of emulsifier by isolate 12 and that a low nutrient concentration enhances production;

Figure Ia illustrates the extracellular production of emulsifier during growth of isolate 12 in Zobell's low nutrient (ZL) medium plus glucose;

Figure 2 is a graph showing the production of emulsifier by isolate 22 in different media and that glucose is required, in addition to a low- nutrient concentration, for the production of emulsifier by isolate 22;

Figure 2a illustrates the extracellular production of emulsifier during growth of isolate 22 in Zobell's low nutrient (ZL) medium plus glucose;

Figure 3 is a graph showing the production of emulsifier by isolate 39 in different media and that glucose is required for the production of emulsifier by isolate 39, and that a high- nutrient concentration minimises the lag in its production so that maximal Emulsification Index (EI0) is achieved 30 hours earlier;

Figure 3a illustrates the extracellular production of emulsifier and surfactant during growth of isolate 39 in Zobell' s high nutrient (ZH) medium plus glucose;

Figure 4 is a graph showing the production of emulsifier by isolate 67 in different media and that glucose, as well as a high nutrient concentration, are both essential for the production of emulsifier by isolate 67;

Figure 4a illustrates the extracellular production of emulsifier and surfactant during growth of isolate 12 in Zobell's high nutrient (ZH) medium plus glucose; Figure 5 illustrates emulsification index 0 to 100%.

The term mutant as used herein is considered to be a bacteria directly derived from a strain of the bacteria of the invention which include at least one mutation to the genome of the bacteria.

The term variant as used herein is considered to be a bacteria which shares the biological pathway of the isolated bacteria which provides for the production of surface active agent.

As described herein the inventors have identified bacterial isolates from the phycosphere of aquatic algae and have extracted a compound or combination of compounds (extract) from the isolates which has or have surface active agent activity. A methodology for identifying the isolates and a methodology for producing the extracts are described. The extract may comprise a single active compound, a combination of compounds, or one or more compounds which have a synergistic effect when found in combination with other compounds in the extract.

Source organisms may be collected from their native environments and isolated.

After the isolation period, samples of the bacteria are grown and selected based on surface active agent activity against selected oils and / or solutions. Surface active activity may be assessed using any suitable assays common in the field. For extraction of the surface active agents, ammonium sulphate precipitation or solvent extraction using isopropanol may be used followed by the use of a tensiometer, e.g. a Nima DST 9005 tensiometer, to detect the production of surfactants, or emulsification assay (see below) to detect the production of emulsifiers. Other methods known in the art could be used to assay for the presence of surfactants.

Emulsifying activity may be determined using any suitable method. For example, measurement of the emulsification index (EI0) may be used to determine the potential of the emulsifiers to form water-in- oil emulsions. Alternatively, spectrophotometry methods may be used to determine if the emulsifiers form oil-in-water emulsions. EIo is defined as a percentage, calculated by dividing the height of the emulsified layer within the oil phase by the total original height of the oil, multiplied by 100 (see figure 13 for an illustration) . The EI0 is measured after vigorously mixing both phases and then allowing the solution to stand for 10 minutes. For spectrophotometry methods an oil and emulsifier solution are mixed together, left to stand for a period of time, e.g. 24 hours, and emulsifying activity measured as the turbidity (or absorbance) at 540nm of the aqueous phase after mildly vortexing the emulsion and allowing it to stand for 60 seconds. Assays as described allow the identification of bacteria which produce surface active activity.

Examples

In order to identify bacterial isolates producing surface active agents, algal species to be studied were selected and suspended in water. To detach the bacteria from the surface of the algae, the suspension was vigorously vortexed and manually shaken.

As an initial sample, 100 isolates were screened by growing each of the isolates in Zobell's liquid medium with glucose and assaying each isolate for emulsifying activity using the method to determine emulsification index (EIo) t or lowering of the surface tension of the culture medium using a tensiometer.

Out of 100 isolates screened, 53 were identified as producing surface active agents.

During growth on glucose, 18 of the isolates expressed surface active agent activity on their cell surface, while 7 isolates released surface- active agents into the culture medium (extracellular) . 03421

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During growth on hydrocarbons, 26 isolates expressed surface-activity on their cell surface, while only 2 produced extracellular surface active agents.

Of the 53 isolates found to exhibit surface activity, 40 of these produced emulsifying activity.

Of the 40 isolates identified as producing emulsifying agents, 4 isolates were determined to produce significant emulsifying activity as when tested with hexadecane.

Table 1

Designated Name of Isolate Emulsification Index (EI0)

For growth in low-nutrient medium: Isolate SAMS 12 100% Isolate SAMS 22 100% For growth in high-nutrient medium: Isolate SAMS 39 100% Isolate SAMS 67 100%

Characterization of the 4 bacterial isolates The four isolates selected from the screening process were further characterised by making dilutions of suspensions of the algae and then plating these dilutions onto marine agar supplemented with hydrocarbons (e.g. hexadecane) . The bacteria that grew out into colonies were picked off the agar plates and then subcultured onto fresh agar plates to obtain pure cultures. Each new isolate was then stored in glycerol at - 8O0C. Growth of each of the four isolates on different carbon sources was determined using GN2 BioLog Microplates. These were inoculated using methods adapted for testing marine organisms, and incubated at 28°C. Growth was noted by visual inspection and indicated as positive (+) , borderline (/) or negative (-) , and examined until a consistent pattern was observed.

Table 2

Carbon source SAMS 12 SAMS 22 SAMS 39 SAMS 67

α-Cyclodextrin + +■ Dextrin + + + + Glycogen + + + + Tween 40 + - + + Tween 80 + - + + N-Acetyl-D- + Galactosamine N-Acetyl-D- - + + - Glucosamine Adonitol + L-Arabinose + — D-Arabitol + + + D-Cellobiose + + + / i-Erythritol + + + D-Fructose / + + L-Fucose + _ D-Galactose / + + Gentiobiose - + + + α-D-Glucose + + + + m-Inositol - - + " + α-D-Lactose + + + - Lactulose - + + - Maltose + + + + D-Mannitol + + + + D-Mannose + + - I D-Melibiose + + - - β-Methyl-D-Glucoside - + + - D-Psicose - - - - D-Raffinose - / - - L-Rhamnose - 4- - - D-Sorbitol - + + + Sucrose - - + + D-Trehalose + / + + Turanose - + + + Xylitol - + - - Pyruvic Acid + - + + Methyl Ester Succinic Acid Mono- + + + + Methyl Ester Acetic Acid + + + + cis-Aconitic Acid - - + + Citric Acid + + + Formic Acid - + + - D-Galactonic - + - - Acid Lactone D-Galacturonic Acid - + + - D-Gluconic Acid - + + + D-Glucosaminic Acid + _ _ D-Glucuromc Acid — + + — α-Hydroxybutyric Acid - + β-Hydroxybutyric Acid - + + + γ-Hydroxybutyric Acid + + / p-Hydroxyphenyl- - - acetic acid Itaconic acid - / + α-Ketobutyric acid + - / - α-Ketoglutaric acid + + / + oi-Ketovaleric acid - - + / D,L-Lactic acid - + + + Malonic acid - + + Propionic acid + + + + Quinic acid - + + + D-Saccharic acid - + - + Sebacic acid - - Succinic acid + + + + Bromosuccinic acid + / + + Succinamic acid + + + / Glucuronamide - / + - L-Alaninamide _ + / + D-Alanine - + + + L-Alanine + + + + L-Alanyl-Glycine + / + - L-Asparagine + + + + L-Aspartic acid + + + - L-Glutamic acid + + + + Glycyl-L-Aspartic acid+ + Glycyl-L-Glutamic acid+ / + L-Histidine - / — + Hydroxy-L-Proline + - + + L-Leucine + + L-Ornithine + L-Phenylalanine + L-Proline + + + + L-Pyroglutam±c acid + / D-Serine + L-Serine + + + + L-Threonine + + + / D,L-Carnitine + / γ-Aminobutyric acid + + + Urocanic acid + Inosine + / + Uridine + + Thymidine + + + Phenylethyl-amine Putrescine + 2-Aminoethanol + + + 2, 3-Butanediol / Glycerol + + + D,L,α-Glycerol + Phosphate α-D-Glucose-1- + Phosphate D-Glucose-6- Phosphate

Extraction of surface active agents In the solvent extraction technique using isopropanol, cells were grown in liquid medium (5 to 10L) in a shaking incubator. At the stage when maximum surface active agent (emulsifying) activity was detected in the medium, the cells were removed by passing the cell culture through a 0.2 μm membrane using a cross-flow tangential filtration system. The filtrate volume was then ultrafiltered through a 100 kDa membrane to concentrate the surface active agent (emulsifying agent) . This was then dialysed with water (5L) and the surface active agents (emulsifying agents) concentrated down to a final volume of ~ IL. Two volumes of isopropanol were then added to the surface active agent solution (emulsifying solution) and stored at 4°C overnight. The precipitated material was then harvested and dried prior to use.

Genetic identification of bacterial isolates Genetic identification was performed on the four isolates to identify them to Genus level (Table 3) . This was performed by 16S rRNA gene seguencing.

Table 3

Isolate No. Genus Source

SAMS 12 Pseudσalteromonas sp. Oban bay shore SAMS 22 Antarctohacter sp. Algal surface SAMS 39 Halomonas sp. Algal surface SAMS 67 Halomonas sp. Algal surface

Optimising Conditions for Emulsifier Production Isolates SAMS 12, 22, 39 and 67 were grown under different culture conditions to determine the most appropriate growth medium leading to the maximum yield of emulsifier. Growth was conducted using different carbon sources (tetradecane, hexadecane, petrol ether, glucose) , and under low or high nutrient concentrations (i.e. organic nitrogen and phosphates) .

The graphs of figures 1, 2, 3 and 4 illustrate the production of emulsifiers by each of the four isolates (SAMS 12, 22, 39 and 67) during growth in different media.

As illustrated by these graphs each isolate produces maximal surface active agent when grown in media particular to the isolate.

Further studies were performed to determine the emulsifying activity, surface tension and cell turbidity when each of the isolates were grown in either Zobell's low nutrient (ZL) or Zobell's high nutrient (ZH) medium plus glucose. The results of these studies are illustrated in the graphs provided in figures Ia, 2a, 3a and 4a.

As illustrated in the graphs of figures Ia and 2a, production of extracellular emulsifier by isolate 12 and isolate 22 respectively commenced during the mid-exponential phase of growth, after 14 hours for isolate 12 and 26 hours for isolate 22, in ZL medium + glucose.

Isolate 12 produced maximum detection levels (EIo - 100%) of emulsifier after only 20 hours, compared with 120 hours for isolate 22. Production of emulsifier by both isolate 12 and isolate 22 was significantly reduced under higher nutrient conditions (ZH medium) , and completely undetectable in the absence of glucose, or when hexadecane or tetradecane are used as an alternative carbon source (results not shown) .

Neither of these isolates appear to produce surfactants since the surface tension did not change over time.

The emulsions formed with hexadecane were stable for many days standing at room temperature.

As illustrated in the graphs in figures 3a and 4a production of extracellular emulsifier by isolate 39 and isolate 67 respectively commenced soon after inoculation in ZH medium + glucose, and reached maximum detection levels (EI0 = 100%) after only 20 hours for isolate 39 and 14 hours for isolate 67.

Both isolates produced surfactant that lowered the surface tension of the medium from 64 to -54 mN/m.

Growth under low nutrient conditions (ZL medium) produced reduced levels of emulsifier, and surfactant production was not detected (results not shown) .

The emulsions formed using hexadecane were stable for many days after standing at room temperature. The following section describes the surface active agents from the bacterial strains SAMS 12, SAMS 22, SAMS 39 and SAMS 67.

Chemical Characterisation of the Emulsifiers Using the methods used to extract surface active agent, for example emulsifiers as exemplified above, extracts from the bacterial cells were obtained.

Identification of sugar components Three equal amounts of each of the emulsifying extracts were dissolved in trifluoroacetic acid (TFA) and hydrolysed for 4 hours at 1000C. The TFA was removed by evaporation and the samples subsequently prepared for analysis by high performance anion exchange chromatography (HPAEC) . The monosaccharide products were separated on a Dionex Carbopac PA-20 column and quantified using external calibration with an equimolar mixture of twelve monosaccharide standards (N-acetylgalactosamine, N-acetylglucosamine, arabinose, rhamnose, fucose, galactose, glucose, mannose, xylose, galacturonic acid, glucuronic acid and muramic acid) that were subjected to acid hydrolysis in parallel with the samples analysed. 1 The results of the monosaccharide analysis are 2 illustrated in Table 4. A high content of uronic 3 acids was determined in all the samples analysed. 4 5 Table 4. Properties of monosaccharide products from 6 acid hydrolysis of the emulsifier extracts produced 7 by NCIMB strains 41242 (SAMS 12), 41245 (SAMS 22), 8 41244 (SAMS 39) and 41243 (SAMS 67) .

0 1 Percent Mean Composition: 2 Component: 3 12 22 39 67 4 5 Rha 1.19 4.90 31.73 3.86 6 Fuc 0.46 16.15 0.00 0.00 7 Gal 0.90 0.48 15.25 1.57 8 GaIN 0.71 1.63 2.46 4.08 9 GIc 10.45 2.96 7.97 6.80 0 GIcN 24.76 31.93 7.70 10.95 1 Man 4.81 9.64 3.27 11.56 2 XyI 27.68 0.00 0.81 0.00 3 Mur 0.31 7.69 0.00 0.13 4 GaIA 23.11 4.27 2.88 2.23 5 GIcA 5.62 20.35 27.92 58.81 6 7 TOTAL (%) : 32.30 15.36 17.32 22.66 fl 9 Standard abbreviations used are: Rha (rhamnose) , Fuc 0 (fucose) , Gal (galactose) , GaIN (galactosamine) , GIc 1 (glucose), GIcN (glucosamine), Man (mannose) , XyI 2 (xylose) , Mur (muramic acid) , GaIA (galacturonic acid) and GIcA (glucuronic acid) . N- acetylglucosamine and N-acetylgalactosamine are de- N-acetylated during the acid hydrolysis and are detected as glucosamine and galactosamine.

Identification of amino acid components Acid hydrolysis and derivatisation was performed on three equal amounts of the emulsifiers extracted from each of the bacterial strains. 10 mg/ml (1% w/v) solutions of each sample were treated in sealed glass vials with 6 N HCl, a crystal of phenol, and flushed with argon. The solutions were heated at 112°C for 22 h, dried thoroughly, and then each sample reconstituted with 10 rtiM HCl. To each sample Waters AccQ. Fluor reagent was added and mixed immediately. The samples were allowed to stand at room temperature for ~1 min and then heated at 550C for 10 min prior to cooling and analysis by HPLC using a Waters Alliance HPLC system equipped with a Zorbax XDB Ci8 reversed phase column equilibrated in AccQ.Tag Eluant A pH5.8 (1 ml/min) . Detection was by fluorescence (λEx = 250 nm; λEm = 395 nm) . Calibration and quantification used co-run hydrolysed amino acid standard mixtures.

The total protein content in the extracts of SAMS 39 and SAMS 67 were found to be the highest of the four extracts, at 26.62% and 40.54%, respectively. The total protein content in the extracts of SAMS 12 and SAMS 22 were comparatively lower at 8.53% and 4.98%, respectively. Table 5. Amino acid composition from acid hydrolysis of the emulsifiers produced by NCIMB strains 41242 (SAMS 12), 41245 (SAMS 22), 41244 (SAMS 39) and 41243 (SAMS 67) .

Percent Mean Composition Component: 12 22 39 67

Asp 1.19 0.68 3.31 7.43 GIu 1.07 0.56 3.68 5.41 Ser 0.50 0.39 1.18 2.31 GIy 0.59 0.42 1.80 2.66 His 0.13 0.08 0.55 0.35 Thr 0.42 0.20 1.72 3.09 Arg 0.48 0.35 1.97 1.68 Ala 0.61 0.43 2.01 3.54 Pro 0.29 0.19 0.98 1.07 Tyr 0.30 0.10 0.97 1.62 Cys 0.31 0.09 0.12 0.04 VaI 0.50 0.36 1.77 2..80 Met 0.25 0.00 0.33 0.12 lie 0.43 0.29 1.39 1..91 Leu 0.63 0.39 2.10 3..25 Lys 0.43 0.20 1.54 1.,26 Phe 0.40 0..25 1.20 2.00

TOTAL(%) : 8.53 4..98 26.62 40.54 Identification of fatty acids Fatty acids were extracted from three equal amounts of the emulsifying extracts by homogenizing 60 mg of emulsifier with 5 volumes of chloroform-.methanol (2:1; v/v) and then adding water to 5% of the final volume. KCl (0.88 %) solution was added to the homogenised material to comprise 25% of final volume. Transesterification to give fatty acid methyl esters (FAMES) were derived using fresh 1% sulfuric acid in methanol and incubating at 1000C for 2 hours. The FAMES were extracted using 5% NaCl and hexane/ether with BHT (1:1). The samples were spotted onto Silica gel G 60 TLC plates and resolved with hexane/ether/glacial acetic acid (80/20/2) alongside FAME standards. FAME spots were identified by spraying dichlorofluorescein solution. The purified FAMES were scraped off and analysed by gas chromatography (GC) using a ZB-WAX (30m x 0.25mm x 0.25μm) column.

No FAMES were identified in any of the four extracts.

Physical Characterisation of the Emulsifiers The emulsifying and stabilizing potential of each of the extracts was tested and compared to that by various commercially-used emulsifiers/stabilizers. The data presented in Tables 6 and 7 were obtained using 0.2 mg/mL of each respective emulsifier dissolved in either 0.1M phosphate-buffered saline (pH 7.4) or 0.1M sodium acetate buffer (pH 3.5), and emulsified with 0.45 mL of a different food oil. The mixture was emulsified using the standard emulsifying assay. The resultant emulsion was allowed to stand for 24 hours before being gently mixed by vortexing for 5 seconds and then allowed to stand for 1 minute, and then the optical density (OD540) of the aqueous phase was measured. All values shown are expressed in units of absorbance as measured at 540 ran.

Table 6 shows the emulsification capability of the four extracts against a range of different food oils when emulsified under neutral pH conditions (pH 7.4) . Except for sesame seed oil, all of the oils were emulsified by at least a factor of 2 by each of the extracts. In particular, sunflower oil was emulsified by at least a factor of 20 by each of the four extracts. The emulsions formed remained stable for many days, and in some cases even months with only less than 10% of the turbidity being reduced due to droplet coalescence. Table 6. Emulsifying activity under neutral conditions (of pH 7.4) of the emulsifiers isolated from each of the four strains (A) against different food oils compared to that by various food emulsifiers/stabilizers (B) .

Emulsifying Activity (OD540) Oil (A) SAMS emulsifiers: None 12 22 39 67

Olive 0.6 2.6 2.1 2.6 2.7 Sunflower 0.1 2.2 2.0 2.5 2.3 Rapeseed 0.9 2.5 2.2 2.5 2.7 Walnut 0.8 2.6 2.1 2.6 2.6 Vegetable 0.4 2.4 2.1 2.5 2.4 Sesame 1.8 2.9 2.2 3.0 2.8 Ground Nut 0.7 2.5 2.3 2.5 2.6

Emulsifying Activity (OD540) Oil (B) Commercial emulsifiers: XG GA Tw80

Olive 1.0 2.5 2.8 Sunflower 0.4 2.3 2.6 Rapeseed 1.4 2.5 2.8 Walnut 1.0 2.1 2.8 Vegetable 0.6 2.1 2.6 Sesame 1.9 2.4 3.2 Ground Nut 0.9 2.4 2.8 The commercial emulsifiers tested were xanthan gum (XG), gum Arabic (GA) and Tween 80 (Tw80) .

Table 7 shows the emulsification capability of the four extracts against a range of different food oils, this time under acidic conditions (pH 3.5) . Except for extract number 67, the three other extracts were capable of significantly emulsifying olive, sunflower, vegetable and ground nut oils. The oils rapeseed, walnut and sesame emulsified relatively highly without the addition of an emulsifier, although in some cases addition of the crude extracts, other than number 67, did enhance the emulsification of these oils slightly. The emulsions formed remained stable for many days, and in some cases even months. Extract 67 appeared to reverse the formation of an emulsion with rapseed and walnut oils under acidic conditions. Table 7. Emulsifying activity under acidic conditions (pH 3.5) of the emulsifiers isolated from each of the four strains (A) against different food oils compared to that of various commercial emulsifiers/stabilizers (B) .

Emulsifying Activity (OD540) Oil (A) SAMS emulsifiers: None 12 22 39 67

Olive 0.3 2.5 2.4 2.5 0.8 Sunflower 0.1 2.5 2.1 2.4 0.3 Rapeseed 2.0 2.6 2.4 2.5 1.2 Walnut 2.5 2.7 2.5 2.5 1.1 Vegetable . 0.2 2.5 2.3 2.5 0.7 Sesame 2.5 2.9 2.7 2.7 2.5 Ground Nut 0.1 2.4 2.3 2.3 0.9

Emulsifying Activity (OD540) Oil (B) Commercial emulsifiers: XG GA Tw80

Olive 2.0 2.3 2.6 Sunflower 2.1 2.2 2.6 Rapeseed 2.0 2.4 2.7 Walnut 2.4 2.6 2.7 Vegetable 2.0 2.2 2.7 Sesame 2.5 2.4 3.1 Ground Nut 0.9 2.1 2.7 The commercial emulsifiers tested were xanthan gum (XG), gum Arabic (GA), and Tween 80 (Tw80) .

The emulsifying and stabilizing potential of each of the extracts was tested following heat treatment (1000C) under acidic (0.1N HCl) and neutral pH conditions, and the data presented in Table 8. Activity was tested using 0.45 mL of olive oil and 0.2 mg/mL of each emulsifier.

Table 8. Effect of heat treatment under acidic neutral pH conditions on the emulsifying activity of the emulsifiers isolated from strains NCIMB 41242 (SAMS 12), 41245 (SAMS 22), 41244 (SAMS 39) and 41243 (SAMS 67) .

Treatment Emulsifying at 1000C Activity (OD540) (min) None 12 22 39 67 in 0.1N HCl: 0 0.1 1.4 0.6 1.9 0.2 5 0.1 0.3 0.3 1.1 0.3 15 0.1 0.3 0.3 1.4 0.5 30 0.1 0.4 0.3 1.4 0.6 60 0.1 0.5 0.3 1.1 2..0 at pH 7.0: 0 0.6 2.5 2.2 2.5 2.,6 5 0.6 2.5 1.4 2.6 1.,9 15 0.6 2.5 1.4 2.6 2..0 30 0.6 2.6 1.5 2.6 2.0 60 0.6 2.5 1.5 2.5 2.0 The ability of the extracts to emulsify and stabilize crude oils and petroleum products was investigated in buffered solution, and in filtered seawater. The solutions were tested for emulsification using 0.45 mL of oil and 0.2 mg/mL of each emulsifier, and the results presented in Tables 9 and 10.

Table 9. Emulsification of different crude oils and petroleum products by each of the four emulsifiers when dissolved in 0. IM phosphate-buffered saline (pH 7.4) .

Crude Oil/ Emulsifying Petroleum Activity (OD540) products None 12 22 39 67 Crude oil Brent blend 0.1 3.7 1.1 3.3 1.7 Beryl 0.1 1.4 1.3 0.9 0.6 Alwyn 0.1 2.3 1.0 2.0 1.9 Other Kerosene 0.0 1.4 1.1 1.5 1.6 Petrol ether 0.0 0.9 0.1 1.4 0.8 Diesel 0.2 1.6 1.0 2.0 1.4 Gasoline 0.0 1.9 0.6 1.6 1.8

As shown in Table 9, all four extracts were able to emulsify a range of crude oils, both light (i.e. Brent and Alwyn) and heavy or viscous types (i.e. Beryl) , as well as some petroleum products under neutral pH conditions. Extracts 12 and 22 produced better emulsions of the heavy variety of crude oil and that were also shown to be more stable. Only extract 39 was most effective at emulsifying petrol ether. All four extracts emulsified kerosene, diesel and gasoline very effectively, except that gasoline was less well emulsified by extract 22.

Table 10. Emulsification of different crude oils and petroleum products by each of the four emulsifiers when dissolved in filtered seawater.

Crude Oil/ Emulsifying Petroleum Activity (OD540) products None 12 22 39 67 Crude oil Brent blend 0.0 2.3 0.7 2.1 2.2 Beryl 0.1 1.0 1.0 0.9 0.8 Alwyn 0.0 1.9 0.7 1.3 1.6 Other Kerosene 0.0 0.9 0 3 1.1 1.4 Petrol ether 0.0 0.6 0.1 0.4 0.7 Diesel 0.1 1.0 0.4 0.7 1.1 Gasoline 0.0 1.6 0.6 0.5 0.9

When dissolved in seawater, all four extracts were found capable of emulsifying all the crude oils and petroleum products tested (Table 10), although at a reduced level compared to 0. IM PBS. Extract 12 was the most effective at emulsifying most of the oils, followed by extracts 67, 39 and 22 respectively.

The emulsifying and stabilizing potential of each of the extracts was tested under cold conditions of 4°C in both neutral (0.1M phosphate-buffered saline at pH 7.4) and acidic (0.1M Na-acetate buffer at pH 3.5) conditions, and following autoclaving at 121°C for 15 minutes in neutral pH (7.4) conditions. The data, presented in Table 11, was obtained using 0.2 mg/mL of each respective emulsifier and activity tested using olive oil. For further comparison, data on emulsification activity at 250C and pH 7.4 is also shown. Table 11. Emulsification of olive oil by each of the four emulsifiers at 40C (under both neutral, pH 7.4, and acidic, pH 3.5, conditions), at 25°C (pH 7.4), and after autoclaving at 121°C for 15 minutes (pH 7.4) .

Emulsifying Activity (OD540) Treatment None 12 22 39 67

At 4°C: pH 7,.4: 0.9 1.9 2-.1 2.6 2.4 pH 3..5: 0.6 1.8 1.9 2,.2 1.3 At 25°C: 0.6 2.7 2.1 2..6 2.7 12:L0C, LSmin: 0.6 2.7 2.1 2.,5 2.1

Anti-microbial Activity Tests The emulsifiers were tested (disc diffusion assay) to evaluate their affects on the growth of four • important pathogens - i.e. Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coll.

The results showed that none of the four emulsifiers had anti-bacterial properties against these pathogens .

All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.