TECHNICAL FIELD

[001] The present invention relates in general to a method of extracting and concentrating natural products from seaweed into a water-based product containing bioactives. This invention has potential but non-limiting uses in improving aquaculture performance of seaweed including and not limited to its use as a growth stimulant for closed loop seaweed culture, reducing microbial outgrowth and fouling in seaweed culture, and antibiotic/antimicrobial activity relating to applications in the cosmetic, agricultural, aquaculture and/or medical industries.

BACKGROUND

[002] Any reference to methods, apparatus or documents of the prior art are not to be taken as consulting any evidence or admission that they formed, or form part of the common general knowledge.

[003] Seaweed are rich in essential vitamins, minerals, proteins, lipids, polysaccharides and enzymes and hence have been used in a variety of industries throughout history. They have been implicated as a vital source in the food, cosmetic, medical, energy and fertilizer industries and also have applications in waste water treatment.

[004] A key aspect of seaweed that distinguishes it from other sources is its abundance i.e. its ability to grow fast at a relatively low cost. This, combined with its ability to produce large amounts of desired oils, starches and the like, and thrive at a wide range of temperature, water quality and nutrient conditions make seaweed a highly desirable entity.

[005] Seaweed extracts have long been used in developing organic fertilizers that aim to improve quality, quantity and yield of various plant types. For example, extracts of Ascophyllum nodosum, a type of brown algae, have been shown to improve the growth of grapes, tomatoes, peppers, potatoes, eggplant and tobacco. Biostimulants from brown seaweed like Phaeophyceae are used in sustainable agricultural applications, and Macrocystis pyrifera and Durvillea potatorum are used in food and industrial applications. [006] The mechanism by which the complex mixture of bioactives in the extract work to improve agriculture is still under extensive research. However, it is important for us to appreciate that the complex mixture of bioactives extracted from different types of seaweed is unique and displays distinct synergistic effects when applied to different plant cultures. This is due to the fact that different plants might be sensitive to different components of a bioactive extract, and the method of extraction utilized can produce different biostimulant mixtures with distinct properties from the same type of seaweed.

[007] The present invention, therefore, provides a method of extraction of volatile bioactives from seaweed that are otherwise undetectable in standard analytical preparations of the dried raw material, providing a unique mixture of chemical constituents that show antimicrobial activity against a range of medically and agriculturally important microbes such as gram negative bacteria, no cytotoxicity effect to human skin cells at cosmetic doses, and enhance aquaculture performance of seaweed when a condensate containing the volatile bioactive mixture extracted is reinoculated into the seaweed culture from which it was extracted.

SUMMARY OF THE INVENTION

[008] In an aspect, the invention is a process for recovering a spectrum of bioactive compounds from seaweed, the process comprising: delivering the seaweed into a drying apparatus; heating a fluidizing gas in a heat exchanger; circulating the heated fluidizing gas through the drying apparatus to remove water and a spectrum of volatile bioactive compounds from the seaweed placed in the drying apparatus; removing water vapor and the spectrum of bioactive compounds from the fluidizing gas by cooling the heated fluidized gas to form a condensate and a substantially dehumidified gas; recovering the condensate comprising condensed water and the spectrum of bioactive compounds. [009] In an embodiment, the seaweed comprises one or more of fresh seaweed, processed seaweed or frozen seaweed.

[010] In an embodiment, the seaweed comprises frozen seaweed and wherein the frozen seaweed is thawed in the drying apparatus.

[011] In an embodiment, the step of circulating the fluidizing gas is carried out over a plurality of drying time intervals and wherein the step of recovering the condensate comprises recovering the condensate at the end of one or more of each said time interval.

[012] In an embodiment, the process further comprises the step of directing the dehumidified gas to the heat exchanger for recirculation through the drying apparatus.

[013] In an embodiment, the heating step comprises heating the fluidizing gas to a temperature in the range of 20°C to 80°C.

[014] In an embodiment, the heating step comprises heating the fluidizing gas to a temperature in the range of 30°C to 50°C.

[015] In an embodiment, the fresh seaweed comprises rhodophytes.

[016] In an embodiment, the seaweed comprises Asparagopsis taxiformis.

[017] In an embodiment the red seaweed is Asparagopsis armata.

[018] In an embodiment, the fluidizing gas is air.

[019] In an embodiment, the process is a closed loop drying process with the drying apparatus comprising a closed loop passage to provide a fluidizing gas loop for recirculating the fluidizing gas repeatedly through the seaweed to progressively remove water vapor and the bioactive compounds over a prolonged time period comprising a plurality of time intervals. [020] In an embodiment, the time period ranges between 3 hours and 72 hours and more preferably between 10 hours and 50 hours and still more preferably between 10 and 20 hours.

[021] In another embodiment, the time period comprises at least 5 hours.

[022] In an embodiment, the recovering step comprises separating and recovering one or more of said bioactive compounds from the condensate.

[023] In an embodiment, the process further comprises the step of regulating the velocity and pressure of the fluidizing gas in the drying apparatus for optimizing removal of the water vapor and the bioactive compounds from the seaweed.

[024] In an embodiment, the step of circulating the fluidizing gas is preceded by placing the seaweed on a plurality of vertically spaced apart drying racks positioned in a drying chamber of the drying apparatus and wherein the step of circulating the heated fluidized gas comprises directing the heated gas through gaps between the drying racks for removing water and the bioactive compounds from the fresh seaweed placed on the drying racks.

[025] In another aspect, the invention provides one or more bioactive compounds produced by the process as described herein.

[026] In yet another aspect, the invention provides a solution concentrate composition derived from seaweed, the composition comprising: one or more haloalkanes with a total relative concentration of at least 1%; one or more haloalcohols with a total relative concentration of at least 3%; one or more haloalkenes with a total relative concentration of at least 3%.

[027] In an embodiment, the composition further comprises one or more haloketones with a total relative concentration of at least 1% and preferably at least 3%.

[028] In an embodiment, the haloketones include tri-bromoacetone or tetra- bromoacetone with a total relative concentration of at least 2%. [029] In an embodiment, one or more halokalkanes include tribromomethane preferably with a relative concentration of at least 10% and more preferably at least 20%.

[030] In an embodiment, one or more haloalkanes include 2-iodopropane preferably with a relative concentration of at least 1% and more preferably at least 3%.

[031] In an embodiment, one or more haloalkanes include di-iodobutane preferably with a relative concentration of at least 2%.

[032] In an embodiment, one or more haloalkanes include dibromoiodomethane preferably with a relative concentration of at least 1% and more preferably at least 3%.

[033] In an embodiment, one or more haloalcohols include 2-iodoethanol preferably with a relative concentration of at least 1% and more preferably at least 3% and more preferably at least 5%.

[034] In an embodiment, one or more haloalcohols further include dibromoethanol with a total relative concentration of at least 1%.

[035] In an embodiment, one or more haloalkenes comprises tribromobutene preferably with a relative concentration of at least 3% and more preferably at least 5%.

[036] In an embodiment, the seaweed from which the composition is derived includes Asparagopsis taxiformis.

[037] In another aspect the invention provides the use of the composition as described herein for producing an antimicrobial agent.

[038] In yet another aspect, the invention provides the use of the composition for producing an antifouling agent.

[039] In another aspect, the invention provides a method of inhibiting growth of bacteria by contacting bacteria with the composition as described herein. [040] In a further aspect, the invention provides an antibacterial composition comprising the composition as described herein for treating, alleviating and/or preventing growth of gram-negative (Gram(-)) bacteria.

[041] In yet another aspect, the invention provides a method of improving growth of seaweed, the method comprising the step of contacting an effective amount of the one or more bioactive compounds in accordance with any one of claims 14 to 24 with a predetermined quantity of the seaweed.

[042] In an embodiment, the method comprises contacting a solution containing the effective amount of the one or more bioactive compounds with the pre-determined quantity of the seaweed.

[043] In an embodiment, the effective amount of the one or more bioactive compounds in the solution ranges between 1% pbw and 10% pbw and more preferably between 1% pbw and 5%pbw.

[044] In an embodiment, the effective amount of the one or more bioactive compounds in the solution is less than at 20% pbw and more preferably between less than 5% pbw.

[045] In an embodiment, the seaweed comprises Asparagopsis taxiformis and/or Asparagopsis armata.

BRIEF DESCRIPTION OF THE DRAWINGS

[046] Preferred features, embodiments and variations of the invention may be discerned from the following detailed description which provides sufficient information for those skilled in the art to perform the invention. The detailed description is not to be regarded as limiting the scope of the preceding summary of the invention in any way. The detailed description will make reference to Figure 1 as follows:

Figure 1 is a box diagram fora process 100 for describing a method of extracting bioactive compounds from seaweed.

Figure 2 shows the chemical structures of the known halomethane compound isomers. Figure 3 shows the chemical structures of the known haloketone compound isomers. Figure 4 shows the chemical structures of the known haloalcohol compound isomers.

Figure 5 shows the chemical structures of the known haloalkane compound isomers.

Figure 6 is a graph showing the various concentration of compounds within the liquid (condensate)-liquid (DCM) extracts. The three panels include initial raw condensate (Figure 6a), three sequential condensate fractions from drying fresh seaweed (Figure 6b) and three sequential condensate fractions from drying frozen seaweed (Figure 6c).

Figure 7 is an example trace of liquid (condensate)-liquid (DCM) extraction using a full- spectrum scan.

Figure 8 shows that results of the Gram (-) bacterial assays. Bacterial growth curves as indicated on panel: Bacteria with no condensate, Combined (combined condensate), Fraction 1, Fraction 2 and Fraction 3, and Control with antibiotic (gentamicin).

Figure 9 shows the results of the Gram (+) bacterial assays. Bacterial growth curves as indicated on panel: Bacteria with no condensate), Combined (combined condensate), Fraction 1, Fraction 2 and Fraction 3, Control with antibiotic (gentamicin).

Figure 10 is a graph showing the final surface area of Asparagopsis with 4 condensate treatments compared to control (n = 5-6 replicate dishes per water treatment).

Figure 11 is a graph showing the specific growth rate of Asparagopsis with 6 condensate fraction treatments compared to control (n = 3-5 replicate jars per water treatment).

Figure 12 shows the in vitro culture of seaweed filaments in various conditions. Treatment of seaweed cultures using condensate (20% inoculation in seawater) versus control treatment (seawater only).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[047] In one or more preferred embodiments, the process or method of extracting bioactive compounds from seaweed can be described as shown in Figure 1.

[048] Figure 1 is a box diagram illustrating a closed loop drying process 100 that utilizes a closed loop drying system 100S in accordance with an embodiment of the present invention. It must be understood that the invention is in no way limited to the specific configuration of the drying system shown in the preferred embodiment and other variations of the drying system as described herein may be used without departing from the spirit and scope of the invention. In the process 100, the air flows in the direction as indicated by the arrows 300, 310, 320, 330, 340 and 350. Fresh fluidizing gas 180 enters the heat exchanger 200 at inlet 205 and gets heated. The hot fluidizing gas 190 then exits the heat exchanger 200 through outlet 210 and passes through the fan 220 that enables the circulation of air within the loop, to enter the drying apparatus 120 at inlet 230. For ease of understanding only one fan 220 has been shown. However, a plurality of fans 220 may be utilized. Within the drying apparatus 120, fresh seaweed 110 is laid on several drying racks 130. The hot fluidizing gas 190 passes over the fresh moisture laden seaweed, drying it and exiting the drying apparatus through outlet 240. The gas now comprises of a mixture of water vapor and a spectrum of volatile bioactive compounds 140. This bioactive mixture 140 then enters a condenser at inlet 250 which condenses the moisture into droplets that are collected as condensate 160 through outlet 260. The dehumidified gas 170 exits the condenser 150 through outlet 270 and gets recycled back into the heat exchanger 200 through inlet 280. Accordingly, the dehumidified gas can once again be directed through drying apparatus by the fan 220 to once again capture moisture and a spectrum of volatile bioactive compounds.

[049] The temperature and pressure of the fluidizing gas been circulated through the system 100S may be regulated depending on the quantity of seaweed and the arrangement of drying racks 130 in the drying apparatus. In one of the trials, a spectrum of bioactive compounds was carried out by producing a condensate from one genus of red seaweed Asparagopsis. Seaweed samples were collected from the field at Moffatt Beach and transported in seawater directly to the facility (~2 hours later). They were labelled based on whether they were collected in deep or shallow water (shallow water representing the intertidal zone). The seaweed was then spun dry in a washing machine and placed on the drying racks 130. The closed loop drying system 100S was used for carrying out closed loop drying of the collected seaweed samples at a low temperature (~40’C) and low humidity for a period of approximately 48 hours. The condensate 160 comprising the condensed water with a spectrum of volatile bioactive compounds from the unit was collected over this same time frame.

[050] Example 1

[051] The foregoing passages describe experimental results in relation to the condensate 160 obtained from the process 100. The condensate is a clear liquid and yielded a pungent smell upon removal of the vessel cap, indicating the presence of volatiles. A method of liquid-liquid partition to analyze the concentration of the volatiles in the water fraction was developed. This method allowed to concentrate the compounds to a point where they could be detected. The liquid-liquid extraction of condensate partitioned the product into dichloromethane (DCM).

[052] Extraction of compounds from the condensed water was conducted by multiple liquid-liquid extractions into DCM. The condensed water (50.0 ± 0.05 ml.) was added to a separatory funnel before the addition of dichloromethane (3.0 ± 0.01 ml_). The dichloromethane was then drained, filtered and vialled. Three liquid-liquid extractions were performed on the one sample to exhaustively extract compounds.

[053] Gas Chromatography-Mass Spectrometry (GC-MS) was performed on a Perkin Elmer Clarus SQ8S fitted with a DB-5 column (Perkin Elmer Elite-5MS, 30.0 m X 0.25 mm, 025 pm). Injections (1.0 pL) were introduced with a 50:1 split ratio with a sample rate of 1.56250 pts/sec. The GC was held at 40.0 °C for 1 min, ramped at 20.0 °C min-1 to 250.0 °C and held for 0 min followed by a 0.5 min equilibration time prior to the next injection. Helium was used as the carrier gas with a flow rate of 1 mL/1 min. Mass spectrometry was performed on a Perkin Elmer Clarus 580 across a weight range of 50 - 340 m/z. Analysis occurred from 3.0 - 12.0 min with a scan rate of 0.3 s. Compounds were identified by referencing mass spectral chromatographs to the NIST library. Relative quantitation was achieved by comparison of peak area ratios (as determined using supplied TurboMass software) of compound to internal standard (equivalent to parts per million or compound (mg)/solvent (L)) which were then evaluated to give compound (g)/algae material (g).

[054] 29 compounds were identified in the condensate of example 1 (peak areas were high enough to quantitate of which 28 halogenated compounds (Table 1) and 1 non- halogenated compound was identified. It may be appreciated that due to limitations of the presently used characterization methods some of the constituent compounds of the condensate may not have been identified. However, other characterization methods may be used for identifying any other compounds that may be present in the condensate 160 in particularly low quantities.

[055] The pH of the condensate from Example 1 was measured to be 3.02 (highly acidic) suggesting the potential for some possible antimicrobial activity (this characteristic has been explored further in example 3). [056] Halogenated compounds of the condensate from example 1 identified as of 16/11/2019 are shown in Table 1 below.

TABLE 1

[057] Table 1 shown above details the number of compounds from example 1 that have been identified as of 16 November 2019. Compounds with two different halogens (e.g. dibromoiodomethane) have been included as a bromoiodo compound and are not counted in either the bromine or iodine column. Compounds that could not be fully elucidated have been attributed in Table 1 according to their most likely substitution (e.g. unknown dibrominated compound placed under ‘Br’ column).

[058] The various compounds identified in example 1 are categorized based on their type and halogenation as shown in Table 2 below. It is important to note that the values shown in Table 2 do not necessarily correlate to the values shown in Table 1 because compounds that could not be correctly identified have been omitted.

TABLE 2

[059] Table 2 shown above details the number of compounds present according to the type of compound and halogenation. Note that ‘type’ does not extend to number of substituents e.g. a dibrominated compound is counted as containing Br while a trichlorodibrominated compound is included only as Cl & Br. Further, compounds that contain two different halogens will be included under the column listing both but will not be present in the columns pertaining to the compounds individually e.g. a dibromochlorinated compound will appear under ‘Br & Cl’ not ‘Br and ‘Cl’ headings. Compounds that could not be fully elucidated were not included in Table 2 as the functional groups were unable to be defined.

[060] The total proportions of halomethanes in the condensate from example 1 are given in Table 3.

TABLE 3

[061] Note that the proportions represent the three halomethanes that were identified in the samples and not concentration. Compounds of known isomers are shown in Figure 2.

[062] The proportions of haloketones present in the condensate of example 1 is presented in Table 4 below.

TABLE 4

[063] The figures represent the four compounds present (all haloacetones). Compounds of known isomers are shown in Figure 3.

[064] The proportions of haloalcohols present in the condensate of example 1 are presented in Table 5 below.

TABLE 5

[065] Compounds of known isomers are portrayed in Figure 4. [066] The proportions of haloalkanes present in condensate of example 1 is presented in Table 6 below.

TABLE 6

[067] Compounds of known isomers are shown in Figure 5.

[068] The proportions of haloalkenes present in the condensate of example 1 is presented in Table 7 below.

TABLE 7

[069] The proportions of haloacids present in the condensate of example 1 is presented in Table 8 below.

TABLE 8

[070] A list of compounds identified in the condensate of example 1 has been summarized along with their respective elution time and Boiling point in Table 9 below. The compound shown below in the column Table 9 is the most likely compound present in the condensate

TABLE 9 [071] As of 16 November 2019, when initial trials were carried out, standards for all compounds present were not obtained to perform accurate quantitation. However, relative peak areas have been used to compare concentrations (which assumes parallel standard curves).

[072] To date, three experiments (on 14/2/2019, 20/2/2019 and 15/11/2019) analyzing compound concentration have been conducted. Totals’ expressed in the Table 10 below represent the sum totals of peak areas determined during multiple extractions; averages of peak areas determined during these experiments have then been determined which are arranged in descending order.

TABLE 10

[073] Comparing the average concentration of compounds in extractions of the condensate at different times indicated a change in the derived composition .

[074] Table 11 below portrays the average values of first extractions only across the experiments and is ordered in descending order.

TABLE 11

[075] In each instance tribromomethane represents the compound of highest concentration. Compound differences may also represent slight differences in storage conditions (i.e. those closest to light sources and heat). Although, storage conditions are very similar and so this would not be expected to influence concentration largely.

[076] Some of the compounds in the condensate, irrespective of the number of extractions used and when they were extracted, were as follows:

1 . tribromomethane (bromoform) 2. 2-iodoethanol

3. tribromobutene

4. 2-iodopropane

5. dibromoiodomethane

6. dibrominated propanol

7. 1 ,1 ,3,3,-tetrabromoacetone

8. dibromobutane

9. 1 ,1 ,3-tribromoacetone

[077] The concentration of compounds within the liquid (condensate)-liquid (DCM) extracts are as shown in Figure 6a. Key compounds as a relative concentration are bromoform, dibromopropanol and 2-iodoethanol.

[078] The concentration of compounds within the plurality of fractions of the liquid (condensate)-liquid (DCM) extracts from fresh seaweed are as shown in Figure 6b. Key compounds as a relative concentration are bromoform, dibrominated propanol, 1 ,1 ,3,3,- tetrabromoacetone, dibromobutane and 1 ,1 ,3-tribromoacetone.

[079] The concentration of compounds within the plurality of fractions of the liquid (condensate)-liquid (DCM) extracts from frozen seaweed are as shown in Figure 6c. Key compounds as a relative concentration are bromoform, dibrominated propanol, and dibromoiodomethane. It was also notable that the higher concentration of bromoform (peak area ratio) across all condensate fractions from frozen material compared to fresh material. Note also the absence of 1 ,1 ,3,3,-tetrabromoacetone, dibromobutane and 1 ,1 ,3- tribromoacetone in condensate fractions from frozen material compared to fresh material. Therefore, the method of separating various fractions allows selective separation and recovery of the constituents.

[080] An example trace of liquid (condensate)-liquid (DCM) extraction using full- spectrum scan is shown in Figure 7. Note that an internal standard was not used in this trace but identification of compounds was achieved by reference to NIST library. Bromoform, the main compound of interest, is also present as the largest peak which is identifiable at 4.50 minutes. [081] Experimental Results (Antimicrobial Activity)

[082] This section relates to the characterization of the antimicrobial activity of the condensate obtained by performing a method in accordance with one embodiment of the present invention using one genus of red seaweed Asparagopsis.

[083] Antimicrobial activity of the condensate and/or fractions was evaluated against a range of relevant microbes.

[084] One of the major components of the condensate is T ribromomethane, also known as bromoform. Previous assays conducted in the laboratory have shown that Bromoform alone is inactive against both Gram(+) and Gram(-) bacteria. This suggests that the other components of the condensate either alone or in combination Tribromomethane may be important in contributing to the results described below.

[085] Liquid broth (LB) cultures of a range of bacteria were used for Gram(+) bacteria: Staphylococcus aureus and Enterococcus faecalis and for Gram(-) bacteria: Escherichia coli, Salmonella enterica and Acinetobacter baumannii.

[086] Single pure colonies were obtained on a nutrient agar plate and grown overnight (up to 24h) in 5ml LB on 37°C,5% C02. 50 ul of the overnight grown pure cultures of each were was added to a fresh 5ml LB broth, and grown for 4.5 hours on 37C with shaking. In 96-well plates, 20 ul of the culture was added to a well up to a total volume of 200ul to several treatments: LB, LB + gentamicin (in total concentration 10 ug), 50ul condensate in LB, 50ul of each fraction in LB. 200ul of LB only was included in each plate as a negative control. Each treatment was assayed in five replicates. The 96-well plate was covered with breathable membrane and incubated for 24 h on 37C with gentle shaking prior to optical density reading. The optical density (OD600) was measured every 20 minutes. Bacterial growth curves in each treatment were analysed by plotting the average (of five replicates) OD600 measurement against time (hours).

[087] Plate cultures of a range of bacteria were developed for Gram(+) bacteria: Staphylococcus aureus and for Gram(-) bacteria: Escherichia coli, Salmonella enterica and Acinetobacter baumannii. [088] Results of the experiments with the 96-well plate assays are as follows: The combined and fractionated condensates were active against all Gram(-) bacteria as shown in Figure 8 below. For combined condensate, a 5-10 hour period of bacteriostatic activity was recorded for Acinetobacter baumannii and Salmonella enterica. For Acinetobacter baumannii, the bacteriostatic effect was primarily observed in Fraction 3, with 24 hours of activity, followed by Fraction 2 with 15-20 hours of bacteriostatic activity and no bacteriostatic activity from Fraction 1. For Salmonella enterica, the bacteriostatic effect was primarily observed in Fraction 2, with 24 hours of activity, followed by Fraction 1 with 15-20 hours of bacteriostatic activity and Fraction 3 and combined condensate with 0-5 hours of bacteriostatic activity. For Escherichia coli, the bacteriostatic effect was primarily observed in Fraction 1 , Fraction 2, and combined condensate with 15-20 hours of activity, and Fraction 3 with 0-5 hours of bacteriostatic activity. For Staphylococcus aureus, there was no clear bacteriostatic effects in any Fraction. These results further demonstrate the unique bioactive profiles of the condensates. For all assayed bacteria, the OD600 readings for Gentamicin (broad spectrum antibiotic) treatments remained constant for the duration of the experiment.

[089] Figure 8 represents Gram(-) bacterial assays. Bacteria as indicated on each panel: Bacteria with no condensate, Combined (combined condensate), Fraction 1 , Fraction 2 and Fraction 3, and Control with antibiotic (gentamicin).

[090] Condensate was not active against the Gram(+) bacteria in the plate assay as shown in Figure 9 for Staphylococcus aureus.

[091] Figure 9 represents Gram(+) bacterial assays. Bacteria with no condensate, Combined (combined condensate), Fraction 1 , Fraction 2 and Fraction 3 and Control with antibiotic (gentamicin).

[092] In addition to 96-well plate assays, screening was done on a range of bacteria using nutrient agar plates for Gram(+) bacteria: Staphylococcus aureus, Streptococcus pyogenes and Enterococcus faecalis and for Gram(-) bacteria: Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae and Acinetobacter baumannii.

[093] Cultures of each were grown in 5ml liquid broth for 3 hours until turbid compared to control. 10Oul of each culture plated and spread on nutrient agar plates. A disk diffusion method was then run using the cultures. Disks were soaked in condensate. A control disk (water) was also used. The culture plates were incubated for 24h and the zone of clearance was recorded.

[094] Results of the experiments are as follows: The condensate was active against all Gram(-) bacteria. A 3-4mm zone of clearance was recorded for Acinetobacter baumannir, a 2-3mm zone of clearance was recorded for Escherichia coli, a 2mm zone of clearance was recorded for Enterobacter cloacae, and a 1 mm zone of clearance was recorded for Pseudomonas aeruginosa. Condensate was not active against any Gram(+) bacteria neither Staphylococcus aureus, Streptococcus pyogenes nor Enterococcus faecalis.

[095] The key finding for this screening is the markedly higher antimicrobial activity for A. baumannii - a nosocomial pathogen which is shaping as a critical clinical problem as it heads towards multidrug resistance.

[096] To check for heterotrophic bacterial presence in the condensate, 10ul (n = 3) of raw, autoclaved and filtered condensate was plated on liquid broth plates. Plates were incubated at 37°C and 5% CO2.

[097] No heterotrophic bacteria were present in the raw condensate raw, nor in the autoclaved or filtered condensate. This indicates that the condensate itself is effectively sterile.

[098] Overall findings relating to antimicrobial activity of condensate (raw/combined) and the relative bioactivity of individual condensate fractions are summarized in Table 12.

Table 12

Experimental Results (Activity against epithelial cells)

[099] This example relates to the characterization of the activity of the condensate, obtained by performing a method in accordance with one embodiment of the present invention using one genus of red seaweed Asparagopsis, against human epithelial cells.

[100] If a cosmetic formulation were to use the condensed water it would likely be in a toner and may be as low as 0.1 % of w/v. We have tested whether the condensed water is toxic to human epithelial cells at this concentration or higher. Cytotoxic and necrotic effects of the “elixir” were measured on the human epithelial cells Hep-2 monolayer in vitro.

[101] Hep-2 seeding was done with 1%, 5% and 10% condensed water (v/v to the Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% Fetal Calf Serum (FCS) media). The survival rate and cell morphology up to 48h or 100% confluency were measured. [102] The raw condensate (above) was compared to filter-sterilized (0.2 urn filter) and autoclaved condensate. We also compared the condensate to dried, ground Asparagopsis from the drying trial (using similar doses). We propagated “survivor cells” into next 48h cycle with addition of the condensate treatments to the media to assess any further differences in activity.

[103] Results of the experiments to check activity against epithelial cells are as follows: Initial investigations of the potential cytopathic/cytotoxic effects of the condensate on the human epithelial cells Hep-2 monolayer was conducted in vitro. Two passages of Hep-2 cells were examined comparing condensate at different concentrations (v/v). First passage (P1) tested viability of Hep-2 cell with 0.1%, 1% and 10% raw condensate (v/v). Dry seaweed and other preparations for the condensate were also investigated. Focusing on the raw condensate at 24 and 48 hours - the 1% condensate treatment was selected for 2nd passage (P2). After 48 hours in P2, all condensate treatments up to 5% contained viable Hep-2 cells. This demonstrates that dilution at a level used in cosmetic formulations are not cytotoxic for human skin cells.

[104] A summary of the treatment conditions and observations described above are provided in Tables 13 and 14 below.

TABLE 13

TABLE 14

Experimental Results (Aquaculture activity and performance)

[105] This example relates to the characterization of the aquaculture performance of seaweed on application of the condensate obtained by performing a method in accordance with one embodiment of the present invention using one genus of red seaweed Asparagopsis. This experiment was performed in two parts. Part one related to testing the effect of low dose condensate treatment on the growth performance of seaweed and part two related to testing the effect of high dose condensate treatment on the culture vessel performance.

[106] Asparagopsis was cultured under controlled environment conditions in plastic petri dishes by excising small growing tips of the sporophyte (filamentous stage) of Asparagopsis. The seaweed was grown in sterilised seawater with f/8 nutrient addition for a culture period of 1 week, at which time seawater was exchanged and a new tip excised. After the cultures were stable (>4 weekly cycles of culture), the condensate was introduced as a treatment (20% inoculation v/v) compared to the control seawater. All other conditions remained the same. Growth, biomass quality and culture vessel surfaces were assessed over time. The trial was repeated twice with condensate.

[107] The protocol of conducting the above described experiment is as follows:

[108] Day 0 - Set up of the experiment was performed as follows:

[109] Water: Filtered seawater (SW) from the ocean was autoclaved. An example composition includes: 2L SW in Schott bottle + 0.2ml of GeO (Germanium dioxide), + 0.05g of powdered cultured enrichment solution (F2 diluted to F/8).

[110] The various condensate (C) concentrations were tested are as follows: Control 0% - 10ml of SW, Condensate 1 % - 9.9ml of SW + 0.1 ml of C, Condensate 5% - 9.5ml of SW + 0.5ml of C, Condensate 10% - 9ml of SW + 1.0ml of C, Condensate 20% - 8ml of SW + 2.0ml of C.

[111] The seaweed Asparagopsis taxiformis was collected from Moffat Beach, Sunshine Coast, Queensland. It was transferred at a later date to be kept in the light cabinet with an average temp of 20.5°C.

[112] The plant room was maintained at an average temperature of 27.5°C, with lights on at 7:20am and lights off at 11 :00pm. The light under shade cloth is at 300-400 Lux.

[113] The experimental set up used for these experiments is as follows: According to treatment, petri dishes (60MM PS STER) were labelled with permanent marker across the lid. 5ml of SWwas added to each petri dish. “C” amount was added in each petri dish as per “Condensate (C) concentrations”. Remaining amount of SW (4.9, 4.5, 4.0 or 3.0 ml) was added to petri dish subsequently. A single filament of seaweed was isolated under the microscope and added to each petri dish. All 54 petri dishes were randomly distributed across 3 white trays, and later placed in the plant room. Four days later, all 3 trays positions were randomly moved to provide a more uniform spread of light flux to dishes. The experiment was run for 2 weeks, with full water change at Day 7. [114] On Day 7, we exchanged the water to all cultures. Half cultures “ND” were put in a new petri dish. Half cultures “SD” were put in same petri dish, after water had been changed.

[115] All new dishes of “ND cultures” were prepared with its specific water treatment prior to sampling. All culture dishes were observed under the dissecting microscope (4. Ox). Some filaments could be found easily with naked eye, but were still checked under the microscope for any small piece of filament still left (sometimes attached) to the petri dish after main filaments were removed. With sterilized (80% ethanol) forceps, followed by a clean seawater dip, seaweed filament was taken from the dish and put in a new dish (“ND” cultures), or taken, water exchanged in the respective dish, and seaweed replaced in the same dish. All 27 petri dishes were randomly distributed across 3 white trays. 24 in total remained (2 not found [likely dead, see notes below], 1 obscured from image analysis).

[116] On Day 14, all 3 trays with petri dishes were taken from the Plant room to the wet lab. Each sample was observed under microscope, taken out of culture, added to a slide, a drop of water was added to it and a cover slip. A photo of each sample was taken under the microscope, all with same settings.

[117] Results of the investigation to observe the effect of low dose condensate treatment to study growth performance of seaweed is as follows: A comparison of growth rates of Asparagopsis cultured in different concentrations of condensate was run over a 2-week period. Final surface area (mm2) of Asparagopsis with 4 condensate treatments compared to control (n = 5-6 replicate dishes per water treatment) were measured. Note pooled sample across new and old dishes.

[118] Key findings, as shown in Figure 10, were that the average surface area of Asparagopsis in 1% was 11.61 mm ² (+/- 2.66 standard error). This was 3-times larger than the control (0% condensate: T-test, p < 0.05). Note that 2-week cultures of small sections of Asparagopsis under higher doses (10% and 20% condensate) led to reduced growth compared to the control.

[119] In addition, a larger scale aquaculture performance trial was run under the same general experimental conditions as above. The experimental set up used for this experiment was as follows: According to treatment, 250ml of SW was added to each culture jar. “F” amount was added in each culture jar as per “Fraction (F) concentrations” of the three sub-fractions of the combined Condensate at either 1% or 5% dose in SW, and a SW control without any dose. A total mass of 0.125 g of seaweed was added to each culture jar. All culture jars were randomly distributed under the light source.

[120] Results of the investigation to observe the effect of low dose condensate fractions (1% and 5%) treatment to study growth performance of seaweed is as follows: A comparison of growth rates of Asparagopsis cultured in different concentrations of condensate fractions (1% and 5%) was run over a 1-week period. On Day 7, all 33 culture jars were harvested. Final weight of Asparagopsis with 6 condensate fraction treatments compared to control (n = 3-5 replicate culture jars per water treatment) were measured.

[121] Key findings at Day 7, as shown in Figure 11 , were that the average growth rate of Asparagopsis in Condensate Fraction 2 at 5% was the highest recorded (7.28% +/- 0.68 standard error). This was on average a significant 60% increase compared to the control (0% condensate fraction, 4.54% +/- 0.68 standard error; T-test, p < 0.05). Condensate Fraction 1 at 5% was the second highest recorded growth rate (5.91% +/- 0.82 standard error). Note that Day 7 results for condensate Fraction 1 at 1% had the lowest recorded growth rate of the condensate treatments (4.67% +/- 1.04 standard error).

[122] There was also anecdotal evidence that some replicates of the 5% condensate (2 out of 6 replicates) treatments contained conspicuously larger volumes of the gland cells (darker bodies inside every cell, bulging cells, arrows), which store the bioactives. The slightly higher dose of condensate may provide an additional benefit of enhancing yields of the bioactives.

[123] Results of investigating the effect of high dose condensate treatment on culture vessel performance in the petri dish experiment is as follows: Cultures of the seaweed had either complete fouling inhibition with 20% condensate or complete (100%) surface fouling of the culture vessels without condensate (0% condensate). Range of different fouling organisms are deterred, seen in green, brown pigmented microbes in culture dishes without condensate (Figure 12).

[124] These findings may be important as a way to naturally sterilize or clean the culture tanks containing the seaweed without killing it. It also reduces the amount of fouling on the seaweed itself. This may be used in tandem with lower dose treatments for growth and bioactive performance over the culture cycle by using the high dose for operational cleaning of tanks without disrupting the beneficial fouling biota.

[125] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. The term “comprises” and its variations, such as “comprising” and “comprised of’ is used throughout in an inclusive sense and not to the exclusion of any additional features.

[126] It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect.

[127] The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted by those skilled in the art.