Eradicating undesired microorganisms in algal aquaculture processes

Abstract

The present approach describes apparatus, systems, methods, and uses for eradicating undesired microorganisms in an algal aquaculture medium. Under the present approach, an algal aquaculture system may employ one or more apparatus of the present approach to eradicate undesired microorganisms in algal aquaculture medium used in the algal aquaculture systems. In some embodiments, apparatus, systems, and methods of the present approach selectively eradicate undesired microorganisms and, if desired, frack the desired algal cells for subsequent harvesting operations.

Description

ERADICATING UNDESIRED MICROORGANISMS IN ALGAL AQUACULTURE PROCESSESFIELD

[0001] The present disclosure relates to apparatus, systems, methods, and uses for eradicating undesired microorganisms in an algal aquaculture process.BACKGROUND

[0002] Algae offer an opportunity as a crop to supply algal oil for renewable fuels and chemicals derived from seawater, atmospheric CO2, sunlight, and nutrients. Algae may be grown on non-arable land for terrestrial crops, which is an additional benefit. One of the key challenges with algal aquaculture to achieve this opportunity is maintaining a practical dominance of the desired algal species while controlling unwanted species. Thus, the problem is to maintain the desired algal species as the dominant species by controlling both competitive and predatory species in the aqueous growth medium. Throughout the last five decades, various companies and researchers have attempted to solve this problem in several ways including: 1) operating algal ponds at extreme conditions of temperature, pH, and salinity; 2) maintaining and deploying large volumes of an expensive inoculum of the desired algae in order to keep its concentration high in the ponds; and 3) using expensive photobioreactors that are closed to the environment, and that exclude potential introduction of unwanted organisms into the aqueous growth medium. Each of these approaches have met with some level of commercial success, but the first approach has been the most successful for larger-scale algal aquaculture of Dunaliella and Spirulina. These two species utilize extreme operating environments of high salinity, and high pH, respectively, to achieve the desired practical dominance of the desired algal species. However, both competitive and predatory species still offer a significant risk to the algal aquaculture with these species. In thespecific case of D. salina, commercial growth conditions are maintained at elevated salinity levels that are unfavorable for the growth of competitive and predatory species. However, this can negatively impact D. salina growth rates and thus the algal productivity per hectare per day.

[0003] Currently, competitive organisms to D. salina are controlled by elevating the salinity of the algal growth medium. This is problematic because the growth rate of D. salina is reduced as the salinity increases. For algal aquaculture, it is also essential that there is an efficient method of harvesting algae. Thus, it would be extraordinarily valuable if a method could be found to reduce the competitive pressure while at the same time maintaining algal growth at a desired rate and harvesting the algae.

[0004] Therefore, current commercial operations must balance growth conditions for the desired species with the conditions needed to maintain a practical dominance of that desired species. Thus, there is a need in commercial algal aquaculture operations for a method to control the practical dominance of the desired algal species using techniques that are unrelated to the algal growth conditions.

[0005] It is an object of this disclosure to describe apparatus, systems, and methods for eradicating (as that term is defined herein) certain undesired microorganisms in an algal aquaculture medium.

[0006] It is also an object of this disclosure to describe algal aquaculture systems employing one or more apparatus of the present approach to eradicate undesired microorganisms in algal aquaculture medium used in the algal aquaculture systems.

[0007] It is also an object of this disclosure to describe apparatus, systems, methods, and uses that selectively eradicate undesired microorganisms and, if desired in specific applications, frack the algal cells for subsequent harvesting processes.SUMMARY

[0008] The present approach describes apparatus, systems, methods, and uses for eradicating undesired microorganisms in an algal aquaculture medium. Under the present approach, an algal aquaculture system may employ one or more apparatus of the present approach to eradicate undesired microorganisms in algal aquaculture medium used in the algal aquaculture systems. In some embodiments, apparatus, systems, methods, and uses of the present approach selectively eradicate undesired microorganisms and, if desired, frack the desired algal cells, e.g., for subsequent harvesting processes.

[0009] Under the present approach, an algal aquaculture medium comprising algae, algal competitors, and / or algal predators, is subjected to sequential pressurization and rapid depressurization, resulting in the efficient destruction of predatory and / or competitive organisms. This “pest control method” may include a pressurization and rapid pressure release, and can be advantageously deployed at one or more locations in an algal aquaculture process. Some embodiments can be accomplished by passing a stream of algal aquaculture medium through a pressure increasing device, such as a pump, followed by a pressure letdown device, such as an orifice or nozzle or expansion.

[0010] Some embodiments of the present approach take the form of methods for eradicating undesired microorganisms in an algal aquaculture medium. The undesired microorganisms may be, e.g., at least one of predators of a microalga of interest and competitors of the microalga of interest. Method embodiments of the present approach include increasing the pressure of the algal aquaculture medium from a first pressure to a second pressure to produce a pressurized algal aquaculture medium at the second pressure at a first outlet, and decreasing the pressure of the pressurized algal aquaculture medium from the second pressure to a third pressureto produce a depressurized algal aquaculture medium at the third pressure. The depressurized algal aquaculture medium contains a decreased quantity of the predators of the microalgae of interest and / or the competitors of the microalga of interest relative to before increasing the pressure and decreasing the pressure. In exemplary embodiments the “decreased quantity” refers to decreased quantity of viable (living) predators and / or competitors of the microalga of interest relative to before increasing the pressure and decreasing the pressure. Viable competitors are able to consume the microalgae of interest, while viable predators are able to consume nutrients that could be used by the microalgae of interest.

[0011] The microalgae of interest may comprise at least one of Dunaliella sp. , Dunaliella salina, Dunaliella kone, and Dunaliella bardawil, and may include combinations thereof. In some embodiments, the microalgae of interest has a cell membrane but not a cell wall.

[0012] The undesired microorganisms may be at least one of Heteramoeba sp., Euplotes sp. , Blepharisma halophila, Cladotricha sigmoidea, Nassula sp. , Parartemia sp. , Fabrea salina, and Artemia salina. One or more of Heteramoeba sp., Euplotes sp., Blepharisma halophila, Cladotricha sigmoidea, Nassula sp., Parartemia sp., Fabrea salina, and Artemia salina can be predators of the microalgae of interest. The undesired microorganisms may be at least one of ciliates, protozoa, amoeba, rotifers, aquatic crustaceans, and Artemia salina. In the present approach, the undesired microorganisms may be one or more competitors of the microalga of interest, and may be one or more of Arthrospira sp., Arthrospira (Spirulina) obliquus, Arthrospira (Spirulina) platensis, Asteromonas sp., Asteromonas gracilis, Cryptomonas sp., Dunaliella sp., Dunaliella bioculata, Dunaliella granulata, Dunaliella maritima, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella polymorpha, Dunaliella primolecta,Dunaliella pseudosalina, Dunaliella quartolecta, Dunaliella terricola, Dunaliella tertiolecta,Dunaliella viridis, Eremosphaera sp., Euglena sp., Euglena gracilis, Euglena viridis, Halospirulina sp., Isochrysis sp., Isochrysis galbana, Isochrysis litoralis, Isochrysis maritima, Lepocinclis sp., Lepocinclis acus, Ochromonas sp., Pavlova sp., Pavlova lutheri, Pavlova salina, and Pavlova viridis.

[0013] In some embodiments, decreasing the pressure occurs within one of less than 1 hour after increasing the pressure, or less than 10 minutes after increasing the pressure, or less than 1 minute after increasing the pressure, or less than 30 seconds after increasing the pressure, or less than 10 seconds after increasing the pressure, or less than 5 seconds after increasing the pressure, or less than 1 second after increasing the pressure. In some embodiments, the residence time may be as low as 1 / 5 s.

[0014] In some embodiments, the depressurized algal aquaculture medium is substantially free of the undesired microorganisms. While there may be undesired microorganism remnants (cellular or body parts) or residue, few - if any - functioning or living undesired organisms will remain in the depressurized algal aquaculture medium. In some embodiments, the decreased quantity of the undesired microorganisms - the quantity remaining after application of the present approach - is less than 50% of an initial quantity of the undesired microorganisms, or 45% of the initial quantity, or 40% of the initial quantity, or 35% of the initial quantity, or 30% of the initial quantity, or 25% of the initial quantity, or 20% of the initial quantity, or 15% of the initial quantity, or 10% of the initial quantity, or 5% of the initial quantity, or 3% of the initial quantity, or 1% of the initial quantity, or 0.5% of the initial quantity. In some embodiments, the depressurized algal aquaculture medium contains less than 50% of an initial quantity of the undesired microorganisms, less than 45% of the initial quantity, less than 40% of the initial quantity, less than 35% of the initial quantity, less than 30% of the initial quantity, less than 25%of the initial quantity, less than 20% of the initial quantity, less than 15% of the initial quantity, less than 10% of the initial quantity, less than 5% of the initial quantity, less than 3% of the initial quantity, less than 1% of the initial quantity, or less than and 0.5% of the initial quantity.

[0015] In some embodiments of the present approach, increasing the pressure of the algal aquaculture medium includes passing the algal aquaculture medium through at least one of a pump, a piston, a compressor, and an injector. It should be appreciated that some embodiments may include more than one pump, piston, compressor, and / or injector. In some embodiments, decreasing the pressure of the pressurized algal aquaculture medium includes passing the algal aquaculture medium through at least one of a nozzle, a valve, an orifice, and an expansion to decrease the pressure of the pressurized algal aquaculture medium. It should be appreciated that some embodiments may include more than one nozzle, valve, orifice, and / or expansion.

[0016] The present approach is suitable for processing of algal aquaculture medium in various scales ranging from a few milliliter laboratory culture to algal aquaculture systems with surface areas extending over several hundreds or even thousands of hectares. The specific benefit of the approach is its scalability enabling processing of large volumes of algal aquaculture medium in a short period of time. The preferred flow rates are at least about 1 m3 / h, or at least about 5 m3 / h, or at least about 15 m3 / h, or at least about 30 m3 / h, or at least about 50 m3 / h, or at least about 100 m3 / h, or at least about 200 m3 / h. In one embodiment the flowrate of the algal aquaculture medium when subjected to increased pressure is greater than about 1 m3 / hour. In one embodiment the flowrate of the algal aquaculture medium when subjected to increased pressure is greater than about 5 m3 / h, greater than about 15 m3 / h, greater than about 30 m3 / h, greater than about 50 m3 / h, greater than about 100 m3 / h, or greater than about 200 m3 / h.

[0017] For some embodiments, the increase from the first pressure to the second pressureis at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar), including increments of 10 psi (0.7 bar) therebetween. For some embodiments, the decrease from the second pressure to the third pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar), including increments of 10 psi (0.7 bar) therebetween. In some embodiments, the pressure increase or decrease may be as high as pressure drops of at least about 725 psi (50 bar), or at least about 1,450 psi (100 bar), or at least about 2,900 psi (200 bar), or at least about 4,351 psi (300 bar), or at least about 5,802 psi (400 bar), or at least about 7,252 psi (500 bar), or at least about 8,702 psi (600 bar), or at least about 10,153 psi (700 bar), or at least about 11,603 psi (800 bar), or at least about 13,053 psi (900 bar), or at least about 14,504 psi (1,000 bar), particularly in embodiments in which partial or complete rupture of D. salina cells is desired. In some embodiments, the difference between the second pressure to the third pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 60 psi (4.1 bar), or at least about 70 psi (4.8 bar), or at least about 80 psi (5.5 bar), or at least about 90 psi (6.2 bar), or at least about 100 psi (6.9 bar), or at least about 110 psi (7.6 bar), or at least about 120 psi (8.3 bar), or at least about 130 psi (9.0 bar), or at least about 140 psi (9.7 bar), or at least about 150 psi (10.3 bar), or at least about 160 psi (11.0 bar), or at least about 170 psi (11.7 bar), or at least about 180 psi (12.4 bar), or at least about 190 psi (13.1 bar), or at least about 200 psi (13.8 bar). For some embodiments, the second pressure andthe third pressure are selected to cause fracking of the microalga of interest. In some embodiments the difference between the second pressure and the third pressure is about 3 - 350 psi, about 3 - 300 psi, about 3 - 250 psi, about 3 - 200 psi, about 3 - 150 psi, about 3 - 100 psi, about 5 - 350 psi, about 5 - 300 psi, about 5 - 250 psi, about 5 - 200 psi, about 5 - 150 psi, about 5 - 100 psi, about 10 - 350 psi, about 10 - 300 psi, about 10 - 250 psi, about 10 - 200 psi, about 10 - 150 psi, about10 - 100 psi, about 15 - 350 psi, about 15 - 300 psi, about 15 - 250 psi, about 15 - 200 psi, about15 - 150 psi, about 15 - 100 psi, about 20 - 350 psi, about 20 - 300 psi, about 20 - 250 psi, about 20 - 200 psi, about 20 - 150 psi, about 20 - 100 psi, about 30 - 350 psi, about 30 - 300 psi, about30 - 250 psi, about 30 - 200 psi, about 30 - 150 psi, about 30 - 100 psi, about 40 - 350 psi, about40 - 300 psi, about 40 - 250 psi, about 40 - 200 psi, about 40 - 150 psi, about 40 - 100 psi, about50 - 350 psi, about 50 - 300 psi, about 50 - 250 psi, about 50 - 200 psi, about 50 - 150 psi, about50 - 100 psi, about 60 - 350 psi, about 60 - 300 psi, about 60 - 250 psi, about 60 - 200 psi, about60 - 150 psi, about 60 - 100 psi, about 70 - 350 psi, about 70 - 300 psi, about 70 - 250 psi, about70 - 200 psi, about 70 - 150 psi, about 70 - 100 psi, about 80 - 350 psi, about 80 - 300 psi, about80 - 250 psi, about 80 - 200 psi, about 80 - 150 psi, or about 80 - 100 psi.

[0018] In some embodiments of the present approach, increasing the first pressure to the second pressure occurs in one of less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

[0019] In some embodiments, decreasing the second pressure to the third pressure occurs in one of less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes,less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

[0020] The present approach may be a continuous process in some embodiments, a semi- continuous process in some embodiments and a batch process for other embodiments. It should be appreciated that some embodiments may be deployed at one or more locations of an algal aquaculture process or facility. For example, the depressurized algal aquaculture medium may be produced at least one of after removing algal aquaculture medium from an algal aquaculture pond, prior to an algal harvesting process, after an algal harvesting process, prior to an algal aquaculture polishing zone, after an algal aquaculture polishing zone, prior to recycling an algal aquaculture medium to an algal aquaculture pond, in an algal aquaculture pond, prior to transferring an algal aquaculture medium from a first algal aquaculture pond to a second algal aquaculture pond, or combinations thereof. In some embodiments, the depressurized algal aquaculture medium is recycled in an algal aquaculture system. In some embodiments, decreasing the pressure to the third pressure destroys less than one of 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0%, of the microalgae of interest in the algal aquaculture medium. In some embodiments of the present approach, the algal aquaculture medium contains about 5 wt% to about 27 wt% salt.

[0021] In some embodiments, the present approach may take the form of an algal aquaculture system having an algal aquaculture pond containing an algal aquaculture medium, the microalgae of interest, and at least one species of undesired microorganism, a first apparatus comprising a first pressurization device configured to increase the pressure of an initial portion of the algal aquaculture medium from a first pressure to a second pressure to produce a pressurized algal aquaculture medium at the second pressure; and a first depressurization device configured todecrease the pressure of the pressurized algal aquaculture medium from the second pressure to a third pressure to produce a depressurized algal aquaculture medium at the third pressure. In such embodiments, the depressurized algal aquaculture medium contains a decreased quantity of the undesired microorganism relative to the initial portion of the algal aquaculture medium.

[0022] Some embodiments may include at least one of a pre-concentration unit, an algal conditioning unit, an algal harvesting unit, and an algal aquaculture medium polishing zone, including combinations thereof. In some embodiments, the first apparatus is configured to receive the initial portion of the algal aquaculture medium from one of the algal aquaculture pond, the preconcentration unit, the algal conditioning unit, the algal harvesting unit, and the algal aquaculture medium polishing zone. In some embodiments, the first apparatus is in fluid communication with one or more selected from the group consisting of the algal aquaculture pond, the pre-concentration unit, the algal conditioning unit, the algal harvesting unit, and the algal aquaculture medium polishing zone, and any combination thereof. The depressurized algal aquaculture medium in some embodiments may be returned to one of the algal aquaculture pond, the pre-concentration unit, the algal conditioning unit, the algal harvesting unit, and the algal aquaculture medium polishing zone. In some embodiments the first apparatus is configured to receive the flowrate of the algal aquaculture medium greater than about 1 m3 / hour, greater than about 5 m3 / h, greater than about 15 m3 / h, greater than about 30 m3 / h, greater than about 50 m3 / h, greater than about 100 m3 / h, or greater than about 200 m3 / h.

[0023] Some embodiments may include a second apparatus having a second pressurization device configured to increase the pressure of a second portion of the algal aquaculture medium from an initial pressure to an increased pressure to produce a second pressurized algal aquaculture medium; and a second depressurization device configured todecrease the pressure of the second pressurized algal aquaculture medium from the increased pressure to a decreased pressure to produce a second depressurized algal aquaculture medium. In such embodiments, the second depressurized algal aquaculture medium contains a decreased quantity of the undesired microorganism relative to the second portion of the algal aquaculture medium. In some embodiments, the second apparatus is configured to receive the second portion of the algal aquaculture medium from one of the algal aquaculture pond, the pre-concentration operation, the algal conditioning operation, the algal harvesting operation, and the algal aquaculture medium polishing zone. In some embodiments, the second depressurized algal aquaculture medium may be returned to one of the algal aquaculture pond, the pre-concentration unit, the algal conditioning unit, the algal harvesting unit, and the algal aquaculture medium polishing zone. Alternatively, or additionally, the second apparatus may be in fluid communication with one or more selected from the group consisting of the algal aquaculture pond, the preconcentration unit, the algal conditioning unit, the algal harvesting unit, the algal aquaculture medium polishing zone, and any combination thereof.

[0024] In some embodiments of the present approach, the first apparatus is configured to decrease the pressure within one of less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second. In some embodiments having a second apparatus, the second apparatus may be configured to decrease the pressure within one of less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, lessthan 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second. In some embodiments, at least one of the depressurized algal aquaculture medium and the second depressurized algal aquaculture medium is substantially free of the undesired microorganisms.

[0025] In embodiments of the present approach, the decreased quantity of the undesired microorganisms - the quantity remaining after operation of the present approach - of at least one of the depressurized algal aquaculture medium and the second depressurized algal aquaculture medium is less than at least one of 50% of an initial quantity of the undesired microorganisms, 45% of the initial quantity, 40% of the initial quantity, 35% of the initial quantity, 30% of the initial quantity, 25% of the initial quantity, 20% of the initial quantity, 15% of the initial quantity, 10% of the initial quantity, 5% of the initial quantity, 3% of the initial quantity, 1% of the initial quantity, and 0.5% of the initial quantity. In embodiments of the present approach, the depressurized algal aquaculture medium and / or the second depressurized algal aquaculture medium contains less than 50% of an initial quantity of the undesired microorganisms, less than 45% of the initial quantity, less than 40% of the initial quantity, less than 35% of the initial quantity, less than 30% of the initial quantity, less than 25% of the initial quantity, less than 20% of the initial quantity, less than 15% of the initial quantity, less than 10% of the initial quantity, less than 5% of the initial quantity, less than 3% of the initial quantity, less than 1% of the initial quantity, or less than and 0.5% of the initial quantity. For example, all undesired microorganisms can be destroyed by the present approach.

[0026] Embodiments may include one or more devices to increase the pressure. For example, the first apparatus may include at least one of a pump, a piston, a compressor, and an injector to increase the pressure of the initial portion algal aquaculture medium. The secondapparatus in some embodiments may include one or more of a pump, a piston, a compressor, and an injector to increase the pressure of the second portion algal aquaculture medium. Embodiments may include one or more devices to decrease the pressure. For example, the first apparatus may comprise at least one of a nozzle, a valve, an orifice, and an expansion, to decrease the pressure of the pressurized algal aquaculture medium. As another example, the second apparatus may comprise at least one of a nozzle, a valve, an orifice, and an expansion, to decrease the pressure of the second pressurized algal aquaculture medium.

[0027] In some embodiments, the increase from the first pressure to the second pressure in the first apparatus may be at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar). In some embodiments, the decrease from the second pressure to the third pressure in the first apparatus may be at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1bar), at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar). In some embodiments, the difference between the second pressure and the third pressure in the first apparatus may be at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 60 psi (4.1 bar), or at least about 70 psi (4.8 bar), or at least about 80 psi (5.5 bar), or at least about 90 psi (6.2 bar), or at least about 100 psi (6.9 bar), or at least about 110 psi (7.6 bar), or at least about 120 psi (8.3 bar), or at least about 130 psi (9.0 bar), or at least about 140 psi (9.7 bar), or at least about 150 psi (10.3 bar), or at least about 160 psi (11.0 bar), or at least about 170 psi (11.7 bar), or atleast about 180 psi (12.4 bar), or at least about 190 psi (13.1 bar), or at least about 200 psi (13.8 bar). In some embodiments, the difference between the second pressure and the third pressure in the first apparatus may be about 3 - 350 psi, about 3 - 300 psi, about 3 - 250 psi, about 3 - 200 psi, about 3 - 150 psi, about 3 - 100 psi, about 5 - 350 psi, about 5 - 300 psi, about 5 - 250 psi, about 5 - 200 psi, about 5 - 150 psi, about 5 - 100 psi, about 10 - 350 psi, about 10 - 300 psi, about 10 - 250 psi, about 10 - 200 psi, about 10 - 150 psi, about 10 - 100 psi, about 15 - 350 psi, about 15 - 300 psi, about 15 - 250 psi, about 15 - 200 psi, about 15 - 150 psi, about 15 - 100 psi, about 20 - 350 psi, about 20 - 300 psi, about 20 - 250 psi, about 20 - 200 psi, about 20 - 150 psi, about 20 -100 psi, about 30 - 350 psi, about 30 - 300 psi, about 30 - 250 psi, about 30 - 200 psi, about 30 -150 psi, about 30 - 100 psi, about 40 - 350 psi, about 40 - 300 psi, about 40 - 250 psi, about 40 -200 psi, about 40 - 150 psi, about 40 - 100 psi, about 50 - 350 psi, about 50 - 300 psi, about 50 -250 psi, about 50 - 200 psi, about 50 - 150 psi, about 50 - 100 psi, about 60 - 350 psi, about 60 -300 psi, about 60 - 250 psi, about 60 - 200 psi, about 60 - 150 psi, about 60 - 100 psi, about 70 -350 psi, about 70 - 300 psi, about 70 - 250 psi, about 70 - 200 psi, about 70 - 150 psi, about 70 -100 psi, about 80 - 350 psi, about 80 - 300 psi, about 80 - 250 psi, about 80 - 200 psi, about 80 -150 psi, or about 80 - 100 psi.

[0028] In some embodiments, the increase from the initial pressure to the increased pressure in the second apparatus may be at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar). The decrease from the increased pressure to the decreased pressure may be at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar),or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar). For some embodiments, the difference between the increased pressure and the decreased pressure in the second apparatus may be at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 60 psi (4.1 bar), or at least about 70 psi (4.8 bar), or at least about 80 psi (5.5 bar), or at least about 90 psi (6.2 bar), or at least about 100 psi (6.9 bar), or at least about 110 psi (7.6 bar), or at least about 120 psi (8.3 bar), or at least about 130 psi (9.0 bar), or at least about 140 psi (9.7 bar), or at least about 150 psi (10.3 bar), or at least about 160 psi (11.0 bar), or at least about 170 psi (11.7 bar), or at least about 180 psi (12.4 bar), or at least about 190 psi (13.1 bar), or at least about 200 psi (13.8 bar). In some embodiments, the difference between the increased pressure and the decreased pressure in the second apparatus may be about 3 - 350 psi, about 3 - 300 psi, about 3 - 250 psi, about 3 - 200 psi, about 3 - 150 psi, about 3 - 100 psi, about 5 - 350 psi, about 5 - 300 psi, about 5 - 250 psi, about 5 - 200 psi, about 5 - 150 psi, about 5 - 100 psi, about 10 - 350 psi, about 10 - 300 psi, about 10 - 250 psi, about 10 - 200 psi, about 10 - 150 psi, about 10 - 100 psi, about 15 - 350 psi, about 15 - 300 psi, about 15 - 250 psi, about 15 - 200 psi, about 15 - 150 psi, about 15 - 100 psi, about 20 - 350 psi, about 20 - 300 psi, about 20 - 250 psi, about 20 - 200 psi, about 20 - 150 psi, about 20 - 100 psi, about 30 - 350 psi, about 30 - 300 psi, about 30 - 250 psi, about 30 - 200 psi, about 30 - 150 psi, about 30 - 100 psi, about 40 - 350 psi, about 40 - 300 psi, about 40 - 250 psi, about 40 - 200 psi, about 40 - 150 psi, about 40 - 100 psi, about 50 - 350 psi, about 50 - 300 psi, about 50 - 250 psi, about 50 - 200 psi, about 50 - 150 psi, about 50 - 100 psi, about 60 - 350 psi, about 60 - 300 psi, about 60 - 250 psi, about 60 - 200 psi, about 60 - 150 psi, about 60 - 100 psi, about 70 - 350 psi, about 70 - 300 psi, about 70 - 250 psi, about 70 - 200 psi, about 70 - 150psi, about 70 - 100 psi, about 80 - 350 psi, about 80 - 300 psi, about 80 - 250 psi, about 80 - 200 psi, about 80 - 150 psi, or about 80 - 100 psi.

[0029] In some embodiments, the first apparatus increases the first pressure to the second pressure in one of less than 1 hour, less than 5 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second. In some embodiments, the first apparatus decreases the second pressure to the third pressure occurs in one of less than 1 hour, less than 5 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second. In some embodiments, the second apparatus increases the initial pressure to the increased pressure in one of less than 1 hour, less than 5 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second. In some embodiments, the second apparatus decreases the increased pressure to the decreased pressure occurs in one of less than 1 hour, less than 5 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

[0030] The present approach may also take the form of a use of a first apparatus having i) a first pressurization device configured to increase the pressure of an initial portion of an algal aquaculture medium from a first pressure to a second pressure to produce a pressurized algal aquaculture medium at the second pressure; and ii) a first depressurization device configured to decrease the pressure of the pressurized algal aquaculture medium from the second pressure to a third pressure to produce a depressurized algal aquaculture medium at the third pressure; for eradicating undesired microorganisms in an algal aquaculture medium in an algal aquaculture system for growing a microalga of interest.

[0031] These and other embodiments will be apparent to the person having an ordinary level of skill in the art in view of this description, the claims appended hereto, and the applicationsincorporated by reference herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 illustrates an apparatus, method, and use according to the present approach.

[0033] Figure 2 depicts an example of an embodiment of the present approach incorporating both a pressurization device and a depressurization device.

[0034] Figure 3 is a diagram of a demonstrative algal aquaculture system and method showing locations where one or more apparatus of the present approach may be installed to eradicate undesired microorganisms.

[0035] Figure 4 shows the results of the impact of pressure drop and recirculation percentage on fracking Dunaliella salina.

[0036] Figure 5 shows photomicrographs illustrating the effect of pressure drop on fracking and rupture of Dunaliella salina algae.DESCRIPTION

[0037] The following description illustrates embodiments of the present approach in sufficient detail to enable practice of the present approach. Although the present approach is described with reference to these specific embodiments, it should be appreciated that the present approach can be embodied in different forms, and this description should not be construed as limiting any appended claims to the specific embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present approach to those skilled in the art. Notwithstanding the foregoing, the following terms are used in the description and claims as having the meanings set forth as follows.

[0038] Algal aquaculture medium: Also referred to as “growth medium” with respect to an algal aquaculture. The algal aquaculture medium is an aqueous solution, such as an aqueous salt solution, for cultivating microalgae, and normally contains competitive species for the microalgae of interest, and / or species that are predators to the microalgae of interest. These species occur naturally. The algal aquaculture medium may contain the microalgae of interest, but in some embodiments it may not. The aqueous solution can be derived from oceans, seas, terminal lakes, aquifers, rivers, wells, underground aquifers, canals, irrigation canals, seawater desalination or evaporation processes, aqueous waste streams, run-offs from agriculture, effluents from an aquaculture facility, other sources of water, and combinations thereof. In some embodiments the algal aquaculture medium includes about 5 wt% to about 27 wt% salt. For example, embodiments of the algal aquaculture medium may contain about 27 wt% salt (measured at 25 °C). Salt is any mixture of ions that can be derived from seawater, and preferably comprises sodium chloride in the presence of other ions that are present in seawater. The salinity of the algal aquaculture medium may comprise any suitable salts for providing the desired salinity. In an embodiment, the salinity comprises sea salts, underground salts, salts of aquifer water, salts of a terminal lake, sodium chloride, and / or any combination of ions present in sea salt. A mixture of sources for the salinity can also be used, in case for example more than one type is readily available. Salinity is a term that defines the total amount of dissolved inorganic solids (salts) in an aqueous solution. The typical salts found in natural waters may include sodium chloride, magnesium chloride, calcium and magnesium sulfates, bicarbonates, and carbonates. It is a standard practice to express salinity as parts per thousand (%o), which is not a true percent but an approximation of the milligrams of salt per gram of water. In more general terms, salinity is indicated by the water source, such as a freshwater, a brackish water, a saline water, and a brine. Ranges of salinity are associated withthese general terms and these ranges are defined as < 0.5 %o (< 0.05 %) for freshwater, 0.5 - 30 %o (0.05 - 3 %) for brackish water, 30 - 50 %o (3 - 5 %) for saline water, and > 50 %o (> 5 %) for a brine. In one embodiment, the algal aquaculture medium comprises salt levels that are elevated above those found in seawater. In one embodiment, the salinity of the algal aquaculture medium is greater than about 5 wt%, such as greater than about 10 wt% or greater than about 15 wt%. In one embodiment, the salinity of the algal aquaculture medium is at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, at least 10 wt%, at least 11 wt%, at least 12 wt%, at least 13 wt%, at least 14 wt%, at least 15 wt%, at least 16 wt%, at least 17 wt%, at least 18 wt%, at least 19 wt%, at least 20 wt%, at least 21 wt%, at least 22 wt%, at least 23 wt%, at least 24 wt%, at least 25 wt%, or at least 26 wt%, up to saturation.

[0039] In some embodiments, the algal aquaculture medium comprises salt levels that are elevated above those found in seawater. In some preferred embodiments, the salt levels are at least 6 wt% NaCl, at least 7 wt% NaCl, at least 8 wt% NaCl, at least 9 wt% NaCl, at least 10 wt% NaCl, at least 11 wt% NaCl, at least 12 wt% NaCl, at least 13 wt% NaCl, at least 14 wt% NaCl, at least 15 wt% NaCl, at least 16 wt% NaCl, at least 17 wt% NaCl, at least 18 wt% NaCl, at least 19 wt% NaCl, at least 20 wt% NaCl, at least 21 wt% NaCl, at least 22 wt% NaCl, at least 23 wt% NaCl, at least 24 wt% NaCl, at least 25 wt% NaCl, or at least 26 wt% NaCl, up to saturation. Often the algal aquaculture medium comprises nutrients (either added or already present) for culturing the algae.

[0040] Weight percent: As used herein, wt % refers to a dry mass of a component in a solution in grams divided by 100 grams of the solution.

[0041] Microalgae: Also referred to in this disclosure using the broader term algae, this refers to the photosynthetic eukaryotic organisms commonly found in freshwater and seawater.The microalgae, such as microalgae of interest, which may be present in an algal aquaculture medium include, but are not limited to, species from the following genera: Acutodesmus, Achnahtes, Amphipora, Amphora, Anabaena, Ankistrodesmus, Arthrospira (also known as Spirulina), Asteromonas, Asterionella, Boekelovia, Borodinella, Botryococcus, Bracteacoccus, Carteria, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Chlorogonium, Chloromonas, Chroomonas, Chrysophaera, Ceratium, Closterium, Coccolithus, Coelastrella, Coscinodiscus, Cosmarium, Cricosphaera, Crocosphaera, Crypthecodinium, Cryptomonas, Cyanocystis, Cyanospira, Cyclotella, Desmodesmus, Ditylum, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Euglena, Fragilaria, Franceia, Galdieria, Gracilaria, Graesiella, Guinardia, Haematococcus, Halocafeteria, Halospirulina, Hantzschia, Hymenomonas, Isochrysis, Lepocinclis, Limnothrix, Micractinium, Microactinium, Microcystis, Monochrysis, Monodus, Monoraphidium, Muriellopsis, Nannochloris, Nannochloropsis, Navicula, Neochloris, Neospongiococcum, Nephrochloris, Nephroselmis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oocystis, Oscillatoria, Ostreococcus, Parachlorella, Pavlova, Peridinium, Phaeodactylum, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Porphyra, Porphyridium, Prochlorococcus, Prototheca, Prymnesium, Pseudanabaena, Pseudochlorella, Pseudochoricystis, Pseudoneochloris, Pyramimonas, Pyrobotrys, Rhodomonas, Scenedesmus, Schizochytrium, Scytonema, Skeletonema, Spirogyra, Stichococcus, Synechococcus, Tetrachlorella, Tetradesmus, Tetraselmis, Thalassiosira, Tisochrysis, Tolypothrix, Tribonema, Trichodesmium, Ulothrix, Vaucheria, Viridiella, Volvox, and genetically-engineered varieties or combinations (mixtures, mixed cultures, co-cultures or synthetic co-cultures) thereof. In exemplary embodiments, the microalgae (such as those mentioned above) have not been genetically modified and do not originate from genetically modified microalgae.

[0042] In one embodiment, the microalga of interest is selected from the group comprising or consisting of Dunaliella sp., Dunaliella bardawil, Dunaliella kone, Dunaliella salina, Dunaliella bioculata, Dunaliella granulata, Dunaliella maritima, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella polymorpha, Dunaliella primolecta, Dunaliella pseudosalina, Dunaliella quartolecta, Dunaliella terricola, Dunaliella tertiolecta, and Dunaliella viridis. In a specific embodiment, the algae is Dunaliella salina. As set forth herein, Dunaliella salina, Dunaliella bardawil, Dunaliella kone, and combinations thereof, are a preferred species for the production of useful and / or valuable components from algal biomass, such as algal oil for renewable fuels and chemicals. In the embodiments and prototypes described herein, Dunaliella salina is the desired algae species.

[0043] Undesired microorganisms: These include predatory microorganisms (or species) and competitive microorganisms (or species), that when present consume and / or inhibit the growth of microalgae species of interest. In preferred embodiments, the present approach targets undesired microorganisms including, but not limited to, Artemia salina (brine shrimp), Fabrea salina, and amoeba. Some embodiments are especially adapted to eradicate Artemia salina and Fabrea salina. For example, the undesired microorganisms in some embodiments may be at least one of Heteramoeba sp., Euplotes sp., Blepharisma halophila, Cladotricha sigmoidea, Nassula sp., Parartemia sp., Fabrea salina, and Artemia salina. As another example, the undesired microorganisms may be at least one of ciliates, protozoa, amoeba, rotifers, aquatic crustaceans, and Artemia salina.

[0044] Predatory microorganisms: Species that are predators to the microalgae or the microalga of interest include, but are not limited to ciliates, protozoa, amoeba, rotifers, aquatic crustaceans, Artemia, and combinations thereof. Predatory species can quickly grow in an algalaquaculture growth medium and consume a large culture of D. salina and other microalga of interest.

[0045] Competitive microorganisms: Competitive (i.e. competitor) microorganisms to the microalgae or microalga of interest comprise organisms that compete with the desired algae for carbon and / or nutrients, such as nitrogen, phosphorus, iron, trace nutrients, and combinations thereof. The competitors may also compete for available light and / or cause shading and thus reduce light availability for the desired algae. The competitors may be algae that do not produce the desired product, or they may be yeasts, fungi, and / or combinations thereof. The competitive microorganisms which may be present in the algal aquaculture growth medium include, but are not limited to, one or more of Arthrospira sp, Arthrospira (Spirulina) obliquus, Arthrospira (Spirulina) platensis, Asteromonas sp., Asteromonas gracilis, Cryptomonas sp., Dunaliella sp., Dunaliella bioculata, Dunaliella granulata, Dunaliella maritima, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella polymorpha, Dunaliella primolecta, Dunaliella pseudosalina, Dunaliella quartolecta, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Eremosphaera sp., Euglena sp., Euglena gracilis, Euglena viridis, Halospirulina sp., Isochrysis sp., Isochrysis galbana, Isochrysis litoralis, Isochrysis maritima, Lepocinclis sp., Lepocinclis acus, Ochromonas sp., Pavlova sp., Pavlova lutheri, Pavlova salina, and Pavlova viridis.

[0046] Eradicate: As used herein, and in the context of destroying undesired microorganisms, the term “eradicate” means to destroy large fractions of the undesired microorganisms present in algal aquaculture medium, such as to destroy at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, of the undesired microorganisms in the algal aquaculture medium. It should be appreciated that thedestroyed fraction will depend on the specific embodiment, and in particular on the magnitude of the pressure change, the fluid dynamics of the system, and the initial volume of the undesired microorganisms.

[0047] The present approach solves the problem of being able to control the population of predatory and / or competitor organisms in a manner that is not associated with the algal growth conditions and that can be used in addition to the growth conditions to maintain a practical dominance of the desired algae. A method for culturing algae in an algal aquaculture system can involve three steps: 1) growing algae in one or more algal aquaculture ponds comprising a growth medium, 2) harvesting the algae, and 3) after harvesting recycling the algal aquaculture medium that is depleted in algae back to an algal aquaculture medium. Thus, it is desirable to have the ability to control the population of predatory organisms in any one of these existing steps to avoid additional capital and operating costs. Specifically for the growth of the alga D. salina, predators include ciliates, protozoa, rotifers, and Artemia. These predators consume D. salina algae, and they can rapidly decimate a culture within days. There is a need to be able to control these specific predators of D. salina besides changing the growth conditions.

[0048] The present approach advantageously uses a rapid pressurization and / or rapid depressurization of a volume of the algal aquaculture medium to eradicate the predominant predatory species and / or competitive species. It should be appreciated that rapid pressurization and / or rapid depressurization may occur on a continuous basis to an algal aquaculture medium flow, or on a batch basis for a batch of algal aquaculture medium.

[0049] Under the present approach, the differential pressure created by rapid pressurization and / or rapid depressurization is used to eradicate the undesirable microorganisms.Unexpectedly, the differential pressure created by the rapid pressurization and / or rapiddepressurization steps needed to achieve destruction of the undesirable microorganisms is surprisingly low, at least about 3 psi (0.2 bar) in some embodiments, or at least about 5 psi (0.34 bar) in some embodiments, or at least about 10 psi (0.69 bar) in some embodiments, or at least about 15 psi (1.0 bar) in some embodiments, or at least about 20 psi (1.4 bar) in some embodiments, or at least about 30 psi (2.1 bar) in some embodiments, or at least about 50 psi (3.4 bar) in some embodiments, or at least about 100 psi (6.9 bar) in some embodiments, or at least about 150 psi (10.3 bar) in some embodiments, as shown in the examples described below.

[0050] Preferably, the pressure change under the present approach is rapid. In some embodiments, the pressure increase occurs in less than 1 hour, and more preferably in less than 10 minutes, and more preferably in less than 1 minute, and may be less than 30 seconds, and may be less than 10 seconds, and may be less than 5 seconds, and may be less than 1 second. In some embodiments, the pressure decrease occurs in less than 1 hour, and more preferably in less than 10 minutes, and more preferably in less than 1 minute, and may be less than 30 seconds, and may be less than 10 seconds, and may be less than 5 seconds, and may be less than 1 second. In some embodiments, the pressure change involves both a pressure increase and a pressure decrease, both of which occur in less than 1 hour, and more preferably in less than 10 minutes, and more preferably in less than 1 minute, and may be less than 30 seconds, and may be less than 10 seconds, and may be less than 5 seconds, and may be less than 1 second. It should be appreciated that the evaluation of ideal times is ongoing, and that the ideal rate of pressure change may vary depending on the embodiment, including factors such as the salinity of the algal aquaculture medium, the undesired microorganism species present, the magnitude of the pressure change, the fluid dynamics of the embodiment, and the methods used to increase and / or decrease pressure. The prototype embodiments described below involved pressure changes of about 50 psi (3.4 bar) overless than 10 seconds, and in some instances less than 5 seconds or less than 1 second.

[0051] It should be appreciated that the rapid pressurization and rapid depressurization steps in the present approach can be performed one time or multiple times to accomplish the objectives of the instant invention. Methods to accomplish multiple pressurization and depressurization steps include, but are not limited to recycling streams around the pressurization and depressurization steps, multiple impellers, multiple stages in a pump system, etc.

[0052] Pressurization of the algal aquaculture medium may occur by any means known in the art, including but not limited to pressure increases generated mechanically, pneumatically, centrifugally, or combinations thereof. Mechanically generated pressurization may be accomplished by, e.g., a pump, a piston, a compressor, an injector, or other methods known in the art, and combinations thereof. It should be appreciated that the pump may be, for example, a multistage centrifugal pump, a gear pump, a double diaphragm pump, pluralities thereof, and combinations thereof. Pneumatically generated pressurization may be accomplished by, e.g., compressing a gas stream and using the elevated pressure to pressurize the algal aquaculture medium. Centrifugal pressurization may be generated by applying a centrifugal force to the algal aquaculture medium such as that experienced in a centrifugal separator, a decanter, a pump impeller, methods to generate cavitation, or other methods known in the art, or combinations thereof. In one embodiment of the present approach, increasing the pressure of the medium comprises passing the medium through at least one of a pump, a piston, a compressor, and an injector.

[0053] Depressurization of the algal aquaculture medium may occur by any means known in the art, including but not limited to pressure reduction generated by passing the fluid into a region of low pressure through, e.g., a nozzle, valve, orifice, expansion, and combinations thereof.Suitable nozzles include nozzle types such as sold by BETE Fog Nozzle Inc., Greenfield, Massachusetts. Examples of orifices include orifices used to measure flow, square orifices, concentric orifice plates, eccentric orifice plates, segmental orifice plates, etc. Suitable expansions include moving the algal aquaculture medium from a region of compression into a lower pressure zone, such as that generated by an impeller blade after it passes or such as that which occurs when the algal aquaculture medium passes from a pipe into a larger region. In one embodiment of the present approach, decreasing the pressure of the medium comprises passing the medium through at least one of a nozzle, a valve, an orifice, and an expansion.

[0054] Figure 1 illustrates an apparatus and method according to an embodiment of the present approach. The apparatus may be temporarily installed in a stream, such as a portable pump and letdown device that may be installed when needed, or in some embodiments may be more of a permanent fixture installed in the desired location. In this flow diagram, algal aquaculture medium 101 flows into apparatus 105. Prior to apparatus 105, algal aquaculture medium 101 is at an initial pressure that is relatively low (e.g., atmospheric pressure or a slightly higher resulting from transfer of algal aquaculture medium 101 to apparatus 105), because most algal cultivation processes use relatively low pressure to transfer algal aquaculture medium throughout various stages, such as paddle wheels and low-head pumps, to avoid unnecessary and expensive equipment. It should be appreciated that algal aquaculture medium 101 may be a flow from various sources in an algal aquaculture process, e.g., an aquaculture pond, an aquaculture conditioning unit, an algal harvesting unit, and the like. It should be appreciated that the term “unit” as used herein refers to one or more apparatus configured to perform the identified process. For example, an algal harvesting unit refers to one or more apparatus configured to perform algal harvesting processes, as are known and used in the art. In some embodiments, algal aquaculture medium 101may flow continuously to apparatus 105, and in other embodiments algal aquaculture medium 101 may be delivered to apparatus 105 in batches.

[0055] Apparatus 105 includes at least one pressurization device, such as those described above. In demonstrative embodiments, the pressurization device is a pump. The pressurization device increases the pressure of the algal aquaculture medium 101 from an initial first pressure to a second pressure higher than the first pressure, to produce a pressurized algal aquaculture medium at the second pressure. In some embodiments, the pressure change resulting from the sudden increase from the first pressure to the second pressure is sufficient to eradicate all or a portion of undesired microorganisms (i.e., at least one of predators and competitors of the microalga of interest) in the algal aquaculture medium 103 exiting apparatus 105. Pressure increases of about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 50 psi (3.4 bar) to about 200 psi (13.8 bar) prove to be effective for eradicating undesired microorganisms. Pressure increases of e.g. as low as 50 psi (3.4 bar) have been found to eradicate large fractions of undesired microorganisms present in algal aquaculture medium, such as at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%. In prototype embodiments, a pressure increase of e.g. 50 psi (3.4 bar) was found to eradicate all undesired microorganisms, as evaluated visually by microscope at a magnification that is sufficient so that damage to the predator or competitor can be seen. Upon inspection of algal aquaculture medium samples treated using prototype embodiments, it appears that the sheer forces acting upon the undesired microorganisms resulting from the rapid pressure increase results in ruptures to the cells of those species (which, generally, can be larger or smaller in size than the microalgae of interest).

[0056] In some embodiments, apparatus 105 may also include at least one depressurization or letdown device, such as those described above. In demonstrative embodiments, the depressurization device is a nozzle, valve, or expansion. The depressurization device decreases the pressure of the pressurized algal aquaculture medium 101 from the second pressure to a third pressure higher than the second pressure, to produce a depressurized algal aquaculture medium at the third pressure. In some embodiments, the pressure change resulting from the sudden decrease from the second pressure to the third pressure is sufficient to eradicate all or a portion of undesired microorganisms in the depressurized algal aquaculture medium. Pressure decreases of e.g. as low as 50 psi (3.4 bar) have been found to eradicate large fractions of undesired microorganisms present in algal aquaculture medium, such as at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or in some embodiments at least 99% resulting in a de minimis remaining amount of undesired microorganisms. In some embodiments, the depressurization eradicates undesired microorganisms that survived pressurization. In prototype embodiments, a pressure decrease of 50 psi (3.4 bar) was found to eradicate all undesired microorganisms, as evaluated visually by microscope at a magnification that was sufficient to view damage to a predator or competitor. In some instances, the degree of magnification used in the microscope was sufficient to view predator species, competitor species, and D. salina algal cells. Upon inspection of algal aquaculture medium samples treated using prototype embodiments, it appears that the rapid pressure decrease results in a rapid expansion of the cell, followed by cell rupture and eradication of the species.

[0057] In some embodiments, the present approach utilizes both a pressurization device and a depressurization device to eradicate undesired microorganisms in an algal aquaculturemedium. Figure 2 depicts an example of an embodiment incorporating both a pressurization device, pump 203, and a depressurization device, orifice 207. As illustrated, algal aquaculture medium 201 enters pump 203 and is pressurized from an initial first pressure to a second pressure. The pressurized algal aquaculture medium 205 then passes through orifice 207 where it is depressurized from the second pressure to a third pressure. Depressurized algal aquaculture medium 209 can be substantially free from undesired microorganisms present in the initial algal aquaculture medium 201.

[0058] The rapid increase and decrease in pressure eradicates all, or substantially all, undesired microorganisms, as evaluated visually by microscope at a magnification that was sufficient to view damage to a predator or competitor. In some instances, the degree of magnification used in the microscope was sufficient to view predator species, competitor species, and D. salina algal cells. Upon inspection of algal aquaculture medium samples treated using prototype embodiments, it appears that the rapid pressure increase and subsequent decrease results in a rapid expansion of the cell, followed by cell rupture and eradication of the undesired microorganisms. It should be appreciated that for some embodiments, a de minimis number of undesired microorganisms may remain following use of the of the present approach. For example, depressurized algal aquaculture medium 209 described above may have a de minimis number of undesired microorganisms remaining, but as discussed above, inspection of samples in prototype embodiments detected no remaining undesired microorganisms. Thus, embodiments of the present approach utilizing both a pressurization device and a depressurization device have been found to eradicate significant fractions of undesired microorganisms present in algal aquaculture medium, such as at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least98%, or in some embodiments at least 99% resulting in a de minimis remaining amount of undesired microorganisms.

[0059] As discussed above, pressure changes of at least about 3 psi (0.2 bar), or at least about 5 psi (0.34 bar), or at least about 10 psi (0.69 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi ( 1.4 bar), or at least about 50 psi (3.4 bar) have been found to eradicate undesired microorganisms in algal aquaculture medium samples. The present approach contemplates higher pressure changes, e.g., as high as about 200 psi (13.8 bar), but prototype embodiments have indicated that pressure changes of e.g. about 3 psi (0.2 bar), 5 psi (0.3 bar), 10 psi (0.7 bar), 15 psi (1.0 bar), 20 psi (1.4 bar), 25 psi (1.7 bar), 30 psi (2.1 bar), 35 psi (2.4 bar), 40 psi (2.8 bar), 45 psi (3.1 bar) or 50 psi (3.4 bar) are sufficient to eradicate all or substantially all of the undesired microorganisms.

[0060] It should be appreciated that the present approach can be incorporated into existing algal aquaculture systems, and at one or more locations within an algal aquaculture system. Figure 3 illustrates a demonstrative algal aquaculture system. It should be appreciated that algal aquaculture systems vary, and that algal aquaculture systems other than as shown in Figure 3 may utilize the present approach. In the illustrated embodiment, an algal aquaculture pond 300 is used for growing a desired algal species (e.g., Dunaliella salina) in an algal aquaculture medium. In preparation for algae harvesting, algal aquaculture medium 301 may be transferred from the algal aquaculture pond 300 to a pre-concentration unit 303 for separating the desired algal species from algal aquaculture medium, or at a minimum increasing the desired algal species concentration. As an example, pre-concentration unit 303 may take the form of a skimming unit in which top-floating algae are skimmed and collected as pre-concentrated algal aquaculture medium 305. Other forms of pre-concentration include, but are not limited to deep-bed filtration, cross-flow microfiltration,centrifugation, flotation, other separation methods known, and combinations thereof. Following the pre-concentration unit 303, pre- concentrated algal aquaculture medium 305 is transported to an algal conditioning unit 307, and algal aquaculture medium is recovered as pre-concentration recycle 304.

[0061] In algal conditioning unit 307, algal cells are fracked to facilitate isolation and subsequent recovery of cellular components and algal biomass. The fracked or “disrupted” or “conditioned” algal biomass is transported as conditioned algal aquaculture medium 309 to an algal harvesting unit 311, and algal aquaculture medium separated during the algal conditioning unit 307 may be recovered as algal conditioning recycle 306. As used herein, a “conditioned” biomass refers to a biomass material that has been treated with one or more conditioning processes before separating from the aqueous stream in a harvesting process. Example conditioning processes include, but are not limited to, fracking, grinding, osmotic shocking, vibratory milling, and the like, as well as other methods described in the art.

[0062] The algal harvesting unit 311 can separate useful and / or valuable components from the algal biomass in conditioned algal aquaculture medium 309, to produce an aqueous algal concentrate 313, and remaining algal aquaculture medium may be recovered as algal aquaculture medium recycle 314. In some embodiments, algal harvesting unit 311 separates algal biomass from water. It should be appreciated that the algal biomass can include one or more of lipids, carotenoids, fatty acids, triacylglycerols, diacylglycerols, monoacylglycerols, oils, chlorophyll, glycerols, phospholipids, carbohydrates, fibers, proteins, among other components.

[0063] Algal aquaculture medium recycle 314, which may or may not include algal aquaculture medium from pre-concentration recycle 304 and / or algal conditioning recycle 306, may be returned to the algal aquaculture pond 300. In some embodiments, all or a portion of algalaquaculture medium recycle 314 may be subjected to a polishing unit 315. One or more polishing processes, such as one or more biological polishing units or polishing zones, can be designed to contain a population of microorganisms that metabolize algae cell residues as well as organic compounds (such as, e.g., glycerol) and / or inorganic compounds released from the microalgae cultivated in the algal aquaculture ponds. Such processes transform algae cell residues, organic compounds, and inorganic compounds into nutrients that the microalgae can utilize. In some embodiments, the polishing process removes residues and other compounds to generate a water stream that may be released or reused. The releasable or reusable water stream (not shown) may be a small portion of the polished algal aquaculture medium. Polished algal aquaculture recycle 319 may be returned to algal aquaculture pond 300, and accumulated compounds may be removed from the system as purged media 320. In some embodiments, Unpolished algal aquaculture medium recycle 317 may be transported to algal aquaculture pond 300. It should be appreciated that makeup water 321 may be added to algal aquaculture pond 300, and that algal aquaculture pond 300 may lose water through natural evaporation 323.

[0064] The present approach may be utilized in one or more of locations 302a-g as illustrated in Figure 3. For example, apparatus 302a may be inserted in makeup water stream 321, and as described above eradicate undesired microorganisms from makeup water stream 321 entering algal aquaculture pond 300. Additionally, or alternatively, apparatus 302b may be inserted in pre-concentration recycle stream 304, and as described above eradicate undesired microorganisms from pre-concentration recycle 304 entering algal aquaculture medium recycle 314. Additionally, or alternatively, apparatus 302c may be inserted in algal conditioning recycle stream 306, to eradicate undesired microorganisms from algal conditioning recycle 306 entering algal aquaculture medium recycle 314. Additionally, or alternatively, apparatus 302d may beinserted in algal aquaculture medium recycle 314, before (as illustrated) or following either or both pre-concentration recycle stream 304 and algal conditioning recycle 306, to eradicate undesired microorganisms entering polishing process 315. Additionally, or alternatively, apparatus 302e may be inserted in unpolished algal aquaculture medium recycle stream 317, to eradicate undesired microorganisms entering algal aquaculture pond 300. Additionally, or alternatively, apparatus 302f may be inserted in polished algal aquaculture medium recycle stream 319, to eradicate undesired microorganisms entering algal aquaculture pond 300. Additionally, or alternatively, apparatus 302g may be inserted in the discharge line from the pond in order to feed any of the preconcentration unit, the algal conditioning unit, and / or the algal harvesting unit to eradicate undesired organisms from entering any of these units. It should be appreciated that apparatus 302a- g may take the form of embodiments described above, including Figures 1 and 2, and may be the same or different.

[0065] Figure 3 illustrates one of many possible algal aquaculture system configurations. Some embodiments of the present approach also provide for recycling used aquaculture medium back to algal aquaculture ponds after harvesting the algae, because 1) less predators and / or competitors will be recycled with the used medium and 2) as the method does not involve addition of chemicals there is no potential of accumulation of harmful chemicals to the aquaculture system even after repeated recycling of the used algal aquaculture medium.

[0066] In some embodiments, the rapid pressurization and / or rapid depressurization effective in eradicating predatory species and competitive species may also be used to frack the cells of the microalga of interest, such as Dunaliella salina. For example, an apparatus according to the present approach may be installed along the pre-concentrated algal aquaculture medium stream 305, or prior to the algal conditioning unit 307, or as part of the algal conditioning unit 307.The apparatus may be configured to effect a pressure increase sufficient to eradicate the undesired microorganisms and also frack the preferred algal species (e.g., Dunaliella salina).

[0067] The fracking processes can be performed on the algal aquaculture medium, preferably during an algal conditioning step before the harvesting process, by subjecting the microalgae to the pressurization and rapid depressurization process. The partial rupturing of algae is referred to as fracking. This partial rupture, or fracking, occurs when the cell membrane is slightly torn or ruptured so that the algal cell retains its initial shape, but the cell membrane is damaged. Fracked algae are preferable to completely ruptured algae due to the difference in size of the resulting particles. Complete rupturing of the algae occurs when the cell membrane is completely ruptured and the internal contents of the algal cell are released and the algal cell does not retain its initial shape. Particles resulting from fracking algae are larger than the particles resulting from the complete rupturing of algae and thus, for example, adsorptive bubble separation processes used for harvesting are more effective when the larger particles are present. Fracking the algae or microalgae can produce fracked cells possessing hydrophobic components while still retaining a significant portion of the intracellular material within the cellular membrane. The hydrophobic nature of the fracked algae allows them to adhere to the bubbles in an adsorptive bubble separation process so that they can efficiently be harvested without the need to add flocculants, collectors, suppressors, or other flotation chemicals that are typically used in dissolved-air flotation. These additives are unwanted because they are costly, contaminate the algal products, and can pose a significant environmental threat.

[0068] The following paragraphs describe prototype embodiments of the present approach.

[0069] In exemplary embodiments, harvesting is carried out in an algal harvesting process(e.g., a harvester apparatus or a harvesting zone) that may include one or more of an adsorptive bubble separation unit, a flotation unit, a centrifugation unit, a flocculation unit, a sedimentation unit, a filtration unit, and / or combinations thereof.

[0070] Figure 4 shows the results of the impact of pressure drop (50 psi (3.4 bar), 125 psi (8.6 bar), and 200 psi (13.8 bar)) and recirculation percentage (50%, 150%, and 350%) on fracking Dunaliella salina. These tests were performed by using a high-pressure pump that obtained the algal aquaculture medium directly from a first algal growth pond that covered about 10 hectares. The algal aquaculture medium was passed through a valve and the pressure was measured between the pump and the valve. After passing through the valve, the algal aquaculture medium was depressurized to atmospheric pressure and discharged to a second algal growth pond. The residence time the algal aquaculture medium was held at the elevated pressure was about 10 seconds. Samples of the algal growth pond were collected from the first algal growth pond and the discharge line from the pump. The number of whole and fracked algal cells were determined using a hemocytometer and microscope. As can be seen, at 50% recirculation, only about 20% of the Dunaliella salina cells were fracked at 200 psi (13.8 bar). However, as recirculation increased the fraction of Dunaliella salina fracked increased. It should be noted that in each evaluated sample, no Artemia salina or Fabrea salina cells were observed to survive the pressure changes. Thus, in embodiments that include fracking the Dunaliella salina cells (or other target species), the apparatus may be configured to operate at higher recirculation and / or at higher pressure changes (e.g., over 200 psi (13.8 bar), to achieve the desired level of fracking.

[0071] In a first example, a culture of Dunaliella salina was grown in a 150-hectare pond in a growth medium that was saturated with NaCl. There was continuous flow of growth medium both into and out of the pond. Neither nitrogen nor phosphorus nutrients were added to the growthmedium. A portion of the growth medium exiting the pond was fed to a multistage centrifugal pump that caused several cycles of pressurization and rapid depressurization of the growth medium. In this medium, the starting concentration of Fabrea salina was 10,000 cells per milliliter, as measured by counting cells on a hemocytometer. The starting concentration of Dunaliella salina was 210,000 cells per milliliter as measured also by counting cells on the hemocytometer. The growth medium flow at 20 liters / minute was subjected to pressurization to 200 psig (13.8 barg) and was depressurized by passing it across a valve to generate an outlet stream at 60 psig (4.1 barg). Thus, the pressure drop across the valve experienced by the growth medium was 140 psi (9.7 bar). Of the Dunaliella salina and Fabrea salina cells fed to the pump and valve system, 90% of the Dunaliella salina cells were fracked, and 100% of the Fabrea salina cells were destroyed - as observed with the microscope. Thus, combining predator control with algal fracking was an efficient method for controlling algal predators. The growth medium was held at the second pressure for less than 10 seconds.

[0072] In a second example, a culture of Dunaliella salina was grown in a 150-hectare pond in a growth medium that was saturated with NaCl. There was continuous flow of growth medium both into and out of the pond. Neither nitrogen nor phosphorus nutrients were added to the growth medium. A portion of the growth medium exiting the pond was fed to a multistage centrifugal pump that caused several cycles of pressurization and rapid depressurization of the growth medium. In this medium, the starting concentration of Fabrea salina was 10,000 cells per milliliter, as measured by counting cells on a hemocytometer. The starting concentration of Dunaliella salina was 20,000 cells per milliliter as measured also by counting cells on the hemocytometer. The growth medium flow at 18 liters / minute was subjected to pressurization to140 psi (9.7 bar) and was depressurized by passing it across a valve to generate an outlet stream at40 psig (2.8 barg). Thus, the pressure drop across the valve experienced by the growth medium was 100 psi (6.9 bar). Of the Dunaliella salina and Fabrea salina cells fed to the pump and valve system, 80% of the Dunaliella salina cells were fracked, and 100% of the Fabrea salina cells were destroyed - as observed with the microscope. Thus, combining predator control with algal fracking was an efficient method for controlling algal predators. The growth medium was held at the second pressure for less than 10 seconds.

[0073] In a third example, a culture of Dunaliella salina was grown in a 1 -hectare pond in a growth medium that comprised 16 wt% NaCl. There was continuous flow of growth medium both into and out of the pond. Both nitrogen and phosphorus nutrients were added to the growth medium. The entirety of the growth medium exiting the pond was fed to a multistage centrifugal pump that caused several cycles of pressurization and rapid depressurization of the growth medium. In this medium, the starting concentration of Fabrea salina was 30,000 cells per milliliter, as measured by counting cells on a hemocytometer. The starting concentration of Dunaliella salina was 870,000 cells per milliliter as measured also by counting cells on the hemocytometer. The growth medium flow of 24 liters / minute was subjected to pressurization to 350 psig (24.1 barg) and was depressurized by passing it across a valve to generate an outlet stream at 30 psig (2.1 barg). Thus, the pressure drop across the valve experienced by the growth medium was 320 psi (22.1 bar). Of the Dunaliella salina and Fabrea salina cells fed to the pump and valve system, 95% of the Dunaliella salina cells were fracked, and 100% of the Fabrea salina cells were destroyed - as observed with the microscope. Thus, combining predator control with algal fracking was an efficient method for controlling algal predators. The growth medium was held at the second pressure for less than 2 seconds.

[0074] In a fourth example, a culture of Dunaliella salina was grown in two 1 -hectareponds operating in parallel with a growth medium that comprised 20 wt% NaCl. There was continuous flow of growth medium both into and out of the pond. Both nitrogen and phosphorus nutrients were added to the growth medium. The entirety of the growth medium exiting the ponds was fed to a multistage centrifugal pump that caused several cycles of pressurization and rapid depressurization of the growth medium. In this medium, the starting concentration of Fabrea salina was 20,000 cells per milliliter, as measured by counting cells on a hemocytometer. The starting concentration of Dunaliella salina was 600,000 cells per milliliter as measured also by counting cells on the hemocytometer. The growth medium flow of 21 liters / minute was subjected to pressurization to 250 psig (17.2 barg) and was depressurized by passing it across a valve to generate an outlet stream at 60 psig (4.1 barg). Thus, the pressure drop across the valve experienced by the growth medium was 190 psi (13.1 bar). Of the Dunaliella salina and Fabrea salina cells fed to the pump and valve system, 85% of the Dunaliella salina cells were fracked, and 100% of the Fabrea salina cells were destroyed - as observed with the microscope. Thus, combining predator control with algal fracking was an efficient method for controlling algal predators. The growth medium was held at the second pressure for less than 1 second.

[0075] In a fifth example, a culture of Dunaliella salina was grown in a 1 -hectare pond operating with a growth medium that comprised 20 wt% NaCl. There was continuous flow of growth medium both into and out of the pond. Both nitrogen and phosphorus nutrients were added to the growth medium. The portion of the growth medium exiting the pond was transferred with a centrifugal pump to a nearby pond that caused one cycle of pressurization and rapid depressurization of the growth medium. In this medium, the starting concentration of Fabrea salina was 40,000 cells per milliliter, as measured by counting cells on a hemocytometer. The starting concentration of Dunaliella salina was 300,000 cells per milliliter as measured also bycounting cells on the hemocytometer. The growth medium flow of 925 liters / minute was subjected to pressurization to 60 psig (4.1 barg) and was depressurized by passing it across a valve to generate an outlet stream at 10 psig (0.7 barg). Thus, the pressure drop across the valve experienced by the growth medium was 50 psi (3.4 bar). Of the Dunaliella salina and Fabrea salina cells fed to the pump and valve system, 20% of the Dunaliella salina cells were fracked, and 100% of the Fabrea salina cells were destroyed - as observed with the microscope. Thus, predator control was achieved by using a centrifugal pump to control algal predators, but some of the Dunaliella salina were damaged in the process. The growth medium was held at the second pressure for less than one second.

[0076] In a sixth example, a culture of Dunaliella salina was grown in a 150-hectare pond in a growth medium that was saturated with NaCl. There was continuous flow of growth medium both into and out of the pond. Neither nitrogen nor phosphorus nutrients were added to the growth medium. A portion of the growth medium exiting the pond was charged to a five-gallon plastic tank. The plastic tank was pressurized to 100 psig (6.9 barg) with air using an air compressor and held for 30 minutes. The growth medium was then discharged from the plastic tank through a tube and a nozzle to the atmosphere. In the growth medium, the starting concentration of Fabrea salina was 10,000 cells per milliliter, as measured by counting cells on a hemocytometer. The starting concentration of Dunaliella salina was 100,000 cells per milliliter as measured also by counting cells on the hemocytometer. The growth medium was subjected to pressurization to 100 psig (6.9 barg) and was depressurized by passing it across a nozzle into a 200-liter vessel. Thus, the pressure drop across the valve experienced by the growth medium was 100 psi (6.9 bar). Of the Dunaliella salina and Fabrea salina cells fed to the pump and valve system, 80% of the Dunaliella salina cells were fracked, and 100% of the Fabrea salina cells were destroyed - as observed with themicroscope. Thus, combining predator control with algal fracking was an efficient method for controlling algal predators. The growth medium was held at the second pressure for at least 30 minutes.

[0077] In a variation of the sixth example, a culture of Dunaliella salina was grown in a 150-hectare pond in a growth medium that was saturated with NaCl. There was continuous flow of growth medium both into and out of the pond. Neither nitrogen nor phosphorus nutrients were added to the growth medium. A portion of the growth medium removed from the pond was fed to a centrifugal pump that pumped the growth medium at 1 liter / minute for one kilometer in a 150mm diameter HDPE pipe before the growth medium was depressurized to atmospheric pressure into another pond. In this medium, the starting concentration of Fabrea salina was 15,000 cells per milliliter, as measured by counting cells on a hemocytometer. The starting concentration of Dunaliella salina was 770,000 cells per milliliter as measured also by counting cells on the hemocytometer. The growth medium was subjected to pressurization to 50 psig / (3.4 barg) and was depressurized by passing it across a valve to generate an outlet stream at atmospheric pressure.

[0078] Thus, the pressure drop across the valve experienced by the growth medium was about 40 psig after frictional losses (2.8 bar). Of the Dunaliella salina and Fabrea salina cells fed to the pump and valve system, 10% of the Dunaliella salina cells were fracked, and 100% of the Fabrea salina cells were destroyed - as observed with the microscope. Thus, combining predator control with algal fracking was an efficient method for controlling algal predators. The residence time of the growth medium at the second pressure was 17 minutes.

[0079] In a seventh example, culture o Artemia sp. was grown in an aerated conical flask at room temperature under a plant light and fed with liquid food suitable for the Artemia. Salinity of the cultivation was maintained at 3.2 weight-%. The growth of Artemia was monitored bymicroscopying samples from the cultivation. A portion of the Artemia culture was charged to a 2.5 L drum. This culture was pressurized by pumping through a tube and a nozzle to the atmosphere. The pressure in the tube before the nozzle was measured. The starting Artemia concentration was 17 living Artemia sp. (adults and nauplii) in a 6 ml sample, as measured by counting the living organisms using a stereomicroscope. There were no dead nor disintegrated body parts of Artemia sp. in the sample. A total of 100 ml of the growth medium was subjected to pressurization to target pressure 7.3 psig (0.5 barg) by a diaphragm pump and was depressurized by passing it through a nozzle to the atmosphere into a plastic beaker. The pressure drop recorded across the nozzle experienced by the growth medium was between 8.7 psi and 22 psi (0.6 bar to 1.5 bar), pumping flow was 42 grams per minute. Triplicate samples of 6 ml were taken from the pumped media, and the average number of living organisms and dead organisms (with some damage and pieces of disintegrated body parts) were calculated with stereomicroscope. Of the initial 17, there were no living Artemia sp., and on average 5 dead Artemia sp. with some damages and 8 pieces of disintegrated body parts observed in the samples. Thus, applying a pressure drop as predator control was an efficient method for controlling algal predators, leaving no living organisms behind.

[0080] Examples 8 to 15 were performed in the same manner as example 7, but with varying pressure drop for the culture. The test parameters for examples 8 to 15 are shown below in Table 1, and the results in Table 2.

[0081] Table 1. Test parameters for examples 8 to 15. Column notations: (A) Example number; (B) Target pressure in psi gauge (upper row) and bar gauge (lower row); (C) Pumping flow in grams per minute; (D) Recorded pressure before nozzle in psi gauge and converted into bar gauge (lower row); (E) Calculated average pressure drop in psi (upper row) and bar (lower row); (F) hold up time at elevated pressure (“target pressure”) in seconds.

[0082] Table 2. Results for Examples 7-15. Column notations: (A) Example number; (B) Target pressure in psi gauge (upper row) and bar gauge (lower row); (C) Average number of Artemia sp. in 6 ml sample, live and intact; (D) Average number of Artemia sp. in 6 ml sample, dead or with some damage; (E) Average number of body parts or pieces of Artemia sp. in 6 ml sample.

[0083] In Table 2, Column C represents the viable Artemia sp., and column D and E represents the non-viable Artemia sp. Examples 8 to 15 show that applying a pressure drop as predator control pursuant to the present approach was an efficient method for controlling algal predators, leaving no viable undesired organisms behind. Examples 7 and 8 show some dead nauplii still detectable, while example 9 to 15 show no whole organisms detectable. It was alsoobserved that the higher the pressure, the smaller the pieces of destroyed Artemia sp. organisms became. Thus, in some embodiments, a higher pressure drop may be used to reduce the size of remaining pieces of destroyed microorganisms.

[0084] In example 16, a culture of Artemia sp. was grown in an aerated conical flask at room temperature under a plant light, and fed with liquid food suitable for the Artemia. Salinity of the cultivation was maintained at 3.2 weight-%. Dunaliella salina was cultivated at 25 °C in a modified 2ASW medium with increased nitrate concentration and salinity of approximately 9 wt- % using plant light and light / dark cycle of 18h / 6h. Artemia cells were collected from their culture medium by sieving and added to the£>. salina culture. In addition, eggs of Artemia sp. were added to the mixture in order to determine if they were impacted by the treatment. The mixed culture was pressurized by pumping through a tube and a nozzle to the atmosphere. The pressure in the tube before the nozzle was measured. The starting Artemia concentration was 3 living Artemia sp. (adults and nauplii) in a 6 ml sample as well as 174 actively moving D. salina cells and 50 immobile D. salina cells in a 10 pl sample, as measured by counting the organisms. There were no dead nor disintegrated body parts of Artemia sp. in the sample. A total of 50 ml of the cell suspension was subjected to pressurization to target pressure 7.3 psig (0.5 barg) by a diaphragm pump and was depressurized by passing it through a nozzle to the atmosphere into a plastic beaker. The pressure drop recorded across the nozzle experienced by the cell suspension was between 5.8 psi and 10 psi (0.4 bar to 0.7 bar), pumping flow was 51 grams per minute. For counting the Artemia sp. cells, triplicate samples of 6 ml were taken from the pumped cell suspension, and the average number of living, dead organisms with some damages, and pieces of disintegrated body parts were calculated. Of the initial 3, there was no living Artemia sp. or any Artemia sp. with small damages present in the sample, while on average 18 pieces of disintegrated body parts wereobserved in the samples. For counting the / ). salina cells, triplicate samples of 10 pl were analyzed with light microscopy and averages of the number of living, actively moving and immobile cells were counted. After the pumping, on average 45 alive and actively moving D. salina cells and 45 immobile D. salina cells were observed. In order to assess the impact of the pumping on the Artemia sp. eggs added to the cell suspension, the number of alive nauplii present in the cell suspension after incubating the sample at room temperature for 5 days, the number of alive nauplii was >100. Thus, applying a pressure drop of between 5.8 psi and 10 psi (0.4 bar to 0.7 bar) as predator control was an efficient method for controlling living algal predators, leaving no living organisms behind, but was not enough to destroy the eggs of the predator. Of the algae still present in the sample after the pumping, 50% were alive and actively moving.

[0085] Examples 17 to 20 were performed in the same manner as example 16, but with varying pressure drop for the culture. The test parameters for example 17 to 20 are shown below in Table 3, and the results for examples 17 to 20 are shown in Table 4

[0086] Table 3. Test parameters for example 17 to 20. Column notations: (A) Example number; (B) Target pressure in psi gauge (upper row) and bar gauge (lower row); (C) Pumping flow in grams per minute; (D) Recorded pressure before nozzle in psi gauge and converted into bar gauge (lower row); (E) Calculated average pressure drop in psi (upper row) and bar (lower row); (F) hold up time at elevated pressure (“target pressure”) in seconds.Table 4. Results for example 17 to 20. Column notations: (A) Example number; (B) Target pressure in psi gauge (upper row) and bar gauge (lower row); (C) Average number of Artemia sp. in 6 ml sample, live and intact; (D) Average number of Artemia sp. in 6 ml sample dead with some damages; (E) Average number of body parts or pieces of Artemia sp. in 6 ml sample; (F) Number of Artemia nauplii after 5 days; (G) Average number of Dunaliella salina alive and actively moving in 10 pl sample; (H) Average number of Dunaliella salina, immobile.

[0087] In Table 4, Column C represents the viable Artemia sp., and column D and E represent the non- viable Artemia sp. Similar to example 7 to 16, examples 17 to 20 also show that applying a pressure drop as predator control was an efficient method for controlling algal predators, leaving no viable undesired organisms behind. The results show that the higher the pressure drop, the smaller the pieces of destroyed Artemia sp. become. The applied pressure drop is also impacting Artemia egg integrity. Example 18 shows that a target pressure drop of 29 psi (2 bar) results in a clear reduction of the integrity of the Artemia eggs, leading to a more than 10 fold reduction of the hatched Artemia nauplii after 5 days, and essentially all Artemia eggs suffered loss of integrity at pressure above that, as shown in examples 19 and 20. Example 18 further shows that after a pressure drop of 29 psi (2 bar), the number of living algae cells is lower compared to the initial but living algae cells are present. Examples 19 and 20 demonstrate that at higher target pressure drops (73 psi / 5.0 bar and 232 psi / 16 bar) the number of living algae cells is low or zero.

[0082] In example 21, a culture of Dunaliella salina were grown in tubular photobioreactors (300) and was preconcentrated (303) with a centrifuge. The algal concentrate was then pumped through a valve and into a receiving vessel at atmospheric pressure. The discharge pressure of the pump was monitored so that the pressure read was the pressure drop experienced by the algal growth medium.

[0088] Photomicrographs of this medium collected before and after the pressure drop were taken and are shown in Figure 5. The algae were approximately 10 microns in diameter, to provide a scale for the images. In Figure 5, two magnification levels are shown for each pressuredrop across the valve. In the highly magnified image, the algal cells are visually intact before they are passed through the pressure drop as shown by the two images collected at 0 bar. They are largely intact or fracked when the pressure drop across the valve was 20, 50, 100, and 200 bar. Above a pressure drop of 300-400 bar, the algal cells are completely ruptured and small pieces of algal biomass can be seen. Pressure drops up to 1,000 bar were evaluated, as can be seen in Figure 5, and effectively result in complete rupture of the microorganisms. Thus, pressure drops of at least about 725 psi (50 bar), or at least about 1450 psi (100 bar), or at least about 2900 psi (200 bar), or at least about 4351 psi (300 bar), or at least about 5802 psi (400 bar), or at least about 7252 psi (500 bar), or at least about 8702 psi (600 bar), or at least about 10,153 psi (700 bar), or at least about 11,603 psi (800 bar), or at least about 13,053 psi (900 bar), or at least about 14,504 psi (1,000 bar), may be utilized in embodiments in which partial or complete rupturing of Dunaliella salina is desired. Dunaliella salina cells have a cell membrane but not a cell wall, so the pressure drop required to rupture this specific alga is lower than that which would be required for an alga that has a cell wall. However, this type of experiment is meant to illustrate how the maximum pressure for a given algal species can be visually determined.

[0089] It should be appreciated that there are locations within most algal aquaculture systems at which fracking or rupturing cells of the microalga of interest (e.g., Dunaliella salina) is undesirable. For example, in some algal aquaculture systems the algal aquaculture medium is transferred between ponds. As another example, it is preferable to retain cells of the microalga of interest (e.g., Dunaliella salina) in make-up water streams and recycled media but minimize or avoid undesired microorganisms. The present approach may be utilized in connection with such transfers, to eradicate undesired microorganisms while having minimal impact on cells of the microalga of interest. As shown in Figure 4, pressure changes of about 50 psi (3.4 bar) have little,if any, impact on the Dunaliella salina cells, but eradicate Artemia salina and Fabrea salina species. It should be appreciated that normal transfer equipment operates at significantly lower pressures and lower pressure change times, for cost-savings and to reduce the likelihood of disturbing microalgal cells such as Dunaliella salina cells. This is evident from the mere presence of the undesired microorganisms in the algal aquaculture medium at various locations in prior art algal aquaculture operations.

[0090] In some embodiments, the present approach may be applied to algal aquaculture medium being recycled. For example, the present approach may be applied to an algal aquaculture media recycle stream. This stream can be, e.g., returning growth media back to the algal aquaculture from the harvesting process, and / or from an algal conditioning process, and / or from a pre-concentration step where algae is optionally skimmed from the top of a pond, and / or from a polishing step. The person having an ordinary level of skill in the art should appreciate that the stream can be coming from a continuous process, a semi-continuous process, or in a batch. The present approach may also be applied to a part of the media recycling stream.

[0091] The present approach may be especially advantageous in algae cultivation pond system streams. In a system with a plurality of interconnected algal aquaculture ponds, the present approach can be applied to a pond that has been heavily impacted by the algal predators by isolating that pond and treating the content of that specific pond. For example, an apparatus such as described above may be used to treat algal aquaculture medium from a single pond. The present approach can also be applied to streams between interconnected ponds, as described above. In these examples, the present approach ensures that undesired microorganisms are not circulated to other ponds, with minimal or negligible impact to Dunaliella salina cells.

[0092] In other embodiments, the present approach may be used in conjunction with othertechniques for controlling microorganism species. After the present approach is used to eradicate undesired microorganisms, and then fracked algae have been harvested, the residual stream can be contacted with oxidizing agents and / or exposed to ultraviolet radiation to reduce the activity of any remaining competing organisms (such as smaller bacteria, archaea and, other algae) before recycling the used aquaculture medium back to the aquaculture ponds. In some embodiments, the residual stream may be fed to a polishing unit in a polishing zone. By first destroying the algal predators and harvesting the fracked algae these additional treatment methods will become more efficient, as larger cells are not disturbing the additional treatment.

[0093] Embodiments of the present approach may also be applied to solar salt evaporation pond systems. In such embodiments, the present approach is advantageous in order to both control the algal competitors and algal predators (e.g., Anemia), and optionally also frack or destroy the algae growing in the ponds.

[0094] In solar salt evaporation pond systems both algae and different competitors and predators may grow that impact the product quality. There are several ways the present invention can be applied in the solar evaporation pond systems, with similar setup as described above in the invention and different embodiments. The target is to control the presence of living organisms and / or adjust the ratio of different microorganisms or species by application of the present approach to flow(s) in / out of the pond system.

[0095] It should be appreciated that the present approach provides a selective method for eradicating microorganisms. In some embodiments the undesired microorganisms are destroyed while leaving the microalgae of interest relatively intact, therefore the ratio of the pest destroyed to the ratio of the algae fracked destroyed is greater than one. In this context, the definition of the“ratio of the pest or algae destroyed” is defined as the concentration of pest or algae ruptured orfracked in the outlet stream to that in the inlet stream.

[0096] In some embodiments, at algal aquaculture media salinities above 15 wt%, the vapor pressure of the algal aquaculture media is lower, and can be as much as 5-40 % lower, than the vapor pressure of fresh water at typical aquaculture temperatures, including but not limited to 5-45 °C. The ratio of the vapor pressure of freshwater to the algal aquaculture medium can be 1.05 - 1.39, at 25 °C, for example. As the examples show, the present approach is also applicable at large flow rates and high salinities.

[0097] The following reference numerals are used in the drawings appended hereto.101 Algal aquaculture medium with undesired microorganisms103 Algal aquaculture medium without or with a reduced number of undesired microorganisms105 Apparatus according to the present approach201 Algal aquaculture medium with undesired microorganisms203 Pressurization device (e.g., pump)205 Pressurized algal aquaculture medium207 Depressurization device (e.g., orifice)209 Depressurized algal aquaculture medium300 Algal aquaculture (e.g., pond)301 Algal aquaculture medium302a-g Potential Apparatus locations303 Pre-concentration process / apparatus304 Pre-concentration recycle305 Pre-concentrated algal aquaculture medium306 Algal conditioning recycle307 Algal conditioning process / apparatus309 Conditioned algal aquaculture medium311 Algal harvesting process / apparatus313 Algal concentrate314 Algal aquaculture medium recycle315 Polishing process / zone / apparatus317 Unpolished algal aquaculture medium recycle319 Polished algal aquaculture medium recycle320 Purged media321 Make-up water323 Evaporated water

[0098] The terminology used in the description of embodiments of the present approach is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the description and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. The present approach encompasses numerous alternatives, modifications, and equivalents as will become apparent from consideration of the following detailed description.

[0099] It will be understood that although the terms “first,” “second,” “third,” “a),” “b),” and “c),” etc. may be used herein to describe various elements of the present approach, and the claims should not be limited by these terms. These terms are only used to distinguish one element of the present approach from another. Thus, a first element discussed below could be termed an element aspect, and similarly, a third without departing from the teachings of the present approach.Thus, the terms “first,” “second,” “third,” “a),” “b),” and “c),” etc. are not intended to necessarily convey a sequence or other hierarchy to the associated elements but are used for identification purposes only. The sequence of operations (or steps) is not limited to the order presented in the claims.

[0100] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling. Also, as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0101] Unless the context indicates otherwise, it is specifically intended that the various features of the present approach described herein can be used in any combination. Moreover, the present approach also contemplates that in some embodiments, any feature or combination of features described with respect to demonstrative embodiments can be excluded or omitted.

[0102] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claim. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0103] The term “about,” as used herein when referring to a measurable value, such as,for example, an amount or concentration and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount. A range provided herein for a measurable value may include any other range and / or individual value therein.

[0104] Having thus described certain embodiments of the present approach, it is to be understood that the scope of the appended claims is not to be limited by particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope thereof as hereinafter claimed.

Claims

CLAIMSWhat is claimed is:

1. A method for eradicating undesired microorganisms in an algal aquaculture medium, the undesired microorganisms being at least one of predators of a microalga of interest and competitors of the microalga of interest, and the method comprising: increasing the pressure of the algal aquaculture medium from a first pressure to a second pressure to produce a pressurized algal aquaculture medium at the second pressure at a first outlet; and decreasing the pressure of the pressurized algal aquaculture medium from the second pressure to a third pressure to produce a depressurized algal aquaculture medium at the third pressure; wherein the depressurized algal aquaculture medium contains a decreased quantity of the at least one of predators of the microalgae of interest and competitors of the microalga of interest relative to before increasing the pressure and decreasing the pressure.

2. The method of claim 1, wherein the salinity of the algal aquaculture medium is greater than about 5 wt% or greater than about 15 wt%.

3. The method of claim 1 or claim 2, wherein the algal aquaculture medium has a flow rate at the first pressure, and the flowrate of the algal aquaculture medium when the first pressure is increased to the second pressure is greater than about 1 m3 / hour, greater than about 5 m3 / h, greater than about 15 m3 / h, greater than about 30 m3 / h, greater than about 50 m3 / h, greater than about 100 m3 / h, or greater than about 200 m3 / h.

4. The method of any one of claims 1 - 3, wherein the ratio of the vapor pressure offreshwater to the algal aquaculture medium is 1.05 - 1.39, optionally at 5 - 45 °C, such as at 25°C.

5. The method of any one of claims 1 - 4, wherein the microalgae of interest has a cell membrane but not a cell wall.

6. The method of any one of claims 1-5, wherein the microalgae of interest comprises at least one member selected from the group consisting of Dunaliella sp. , Dunaliella salina, Dunaliella kone, and Dunaliella bardawil.

7. The method of any one of claims 1-6, wherein the undesired microorganisms comprise at least one member selected from the group consisting of Heteramoeba sp. , Euplotes sp. , Blepharisma halophila, Cladotricha sigmoidea, Nassula sp. , Parartemia sp. , Fabrea salina, and Artemia salina.

8. The method of any one of claims 1-7, wherein the undesired microorganisms comprise at least one member selected from the group consisting of ciliates, protozoa, amoeba, rotifers, aquatic crustaceans, and Artemia salina.

9. The method of any one of claims 1-8, wherein decreasing the pressure occurs within one of less than 1 hour after increasing the pressure, less than 10 minutes after increasing the pressure, less than 1 minute after increasing the pressure, less than 30 seconds after increasing the pressure, less than 10 seconds after increasing the pressure, less than 5 seconds after increasing the pressure, and less than 1 second after increasing the pressure.

10. The method of any one of claims 1-9, wherein the depressurized algal aquaculture medium is substantially free of the undesired microorganisms.

11. The method of any one of claims 1-10, wherein the depressurized algal aquaculture medium contains less than 50% of an initial quantity of the undesired microorganisms,less than 45% of the initial quantity, less than 40% of the initial quantity, less than 35% of the initial quantity, less than 30% of the initial quantity, less than 25% of the initial quantity, less than 20% of the initial quantity, less than 15% of the initial quantity, less than 10% of the initial quantity, less than 5% of the initial quantity, less than 3% of the initial quantity, less than 1% of the initial quantity, or less than 0.5% of the initial quantity.

12. The method of any one of claims 1-11, wherein increasing the pressure of the algal aquaculture medium comprises passing the algal aquaculture medium through at least one of a pump, a piston, a compressor, and an injector.

13. The method of any one of claims 1-12, wherein decreasing the pressure of the pressurized algal aquaculture medium comprises passing the algal aquaculture medium through at least one of a nozzle, a valve, an orifice, and an expansion to decrease the pressure of the pressurized algal aquaculture medium.

14. The method of any one of claims 1-13, wherein the competitors of the microalga of interest comprise at least one member selected from the group consisting of: Arthrospira sp., Arthrospira (Spirulina) obliquus, Arthrospira (Spirulina) platensis, Asteromonas sp., Asteromonas gracilis, Cryptomonas sp., Dunaliella sp., Dunaliella bioculata, Dunaliella granulata, Dunaliella maritima, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella polymorpha, Dunaliella primolecta, Dunaliella pseudosalina, Dunaliella quartolecta, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Eremosphaera sp., Euglena sp., Euglena gracilis, Euglena viridis, Halospirulina sp., Isochrysis sp., Isochrysis galbana, Isochrysis litoralis, Isochrysis maritima, Lepocinclis sp., Lepocinclis acus, Ochromonas sp., Pavlova sp., Pavlova lutheri, Pavlova salina, and Pavlova viridis.

15. The method of any one of claims 1-14, wherein the increase from the first pressureto the second pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.3 bar), at least about 10 psi (0.7 bar), at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar), or at least about 725 psi (50 bar), or at least about 1450 psi (100 bar), or at least about 2900 psi (200 bar), or at least about 4351 psi (300 bar), or at least about 5802 psi (400 bar), or at least about 7252 psi (500 bar), or at least about 8702 psi (600 bar), or at least about 10,153 psi (700 bar), or at least about 11,603 psi (800 bar), or at least about 13,053 psi (900 bar), or at least about 14,504 psi (1,000 bar).

16. The method of any one of claims 1-15, wherein the decrease from the second pressure to the third pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.3 bar), or at least about 10 psi (0.7 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar).

17. The method of any one of claims 1-16, wherein the difference between the second pressure to the third pressure is at least about 3 psi (0.2 bar), or at least about 5 psi, or at least about10 psi (0.7 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 60 psi (4.1 bar), or at least about70 psi (4.8 bar), or at least about 80 psi (5.5 bar), or at least about 90 psi (6.2 bar), or at least about100 psi (6.9 bar), or at least about 110 psi (7.6 bar), or at least about 120 psi (8.3 bar), or at least about 130 psi (9.0 bar), or at least about 140 psi (9.7 bar), or at least about 150 psi (10.3 bar), or at least about 160 psi (11.0 bar), or at least about 170 psi (11.7 bar), or at least about 180 psi (12.4 bar), or at least about 190 psi (13.1 bar), or at least about 200 psi (13.8 bar).

18. The method of any one of claims 1-17, wherein the algal aquaculture medium has a flow rate at the first pressure, and the flow rate of the algal aquaculture medium when the firstpressure is increased to the second pressure is greater than about 1 m3 / hour or greater than about 5 m3 / hour, and the decrease from the second pressure to the third pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.3 bar), or at least about 10 psi (0.7 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2. 1 bar), or at least about 50 psi (3.4 bar), or at least about 60 psi (4. 1 bar), or at least about 70 psi (4.8 bar), or at least about 80 psi (5.5 bar), or at least about 90 psi (6.2 bar), or at least about 100 psi (6.9 bar), or at least about 110 psi (7.6 bar), or at least about 120 psi (8.3 bar), or at least about 130 psi (9.0 bar), or at least about 140 psi (9.7 bar), or at least about 150 psi (10.3 bar), or at least about 160 psi (11.0 bar), or at least about 170 psi (11.7 bar), or at least about 180 psi (12.4 bar), or at least about 190 psi (13.1 bar), or at least about 200 psi (13.8 bar).

19. The method of any one of claims 1-18, wherein increasing the first pressure to the second pressure occurs in one of less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

20. The method of any one of claims 1-19, wherein decreasing the second pressure to the third pressure occurs in one of less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

21. The method of any one of claims 1 -20, wherein the method is a continuous or semi- continuous process.

22. The method of any one of claims 1-21, wherein the method is a batch process.

23. The method of any one of claims 1 -22, wherein the depressurized algal aquaculture medium is produced at least one of after removing algal aquaculture medium from an algal aquaculture pond, prior to an algal harvesting process, after an algal harvesting process, prior to an algal aquaculture polishing zone, after an algal aquaculture polishing zone, prior to recycling an algal aquaculture medium to an algal aquaculture pond, in an algal aquaculture pond, and prior to transferring an algal aquaculture medium from a first algal aquaculture pond to a second algal aquaculture pond.

24. The method of any one of claims 1-23, wherein the second pressure and the third pressure are selected to cause fracking of the microalga of interest.

25. The method of any one of claims 1 -24, wherein the depressurized algal aquaculture medium is recycled in an algal aquaculture system.

26. The method of any one of claims 1-25, wherein the decreasing the pressure to the third pressure destroys less than one of 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0%, of the microalgae of interest in the algal aquaculture medium.

27. The method of any one of claims 1-26, wherein the algal aquaculture medium contains about 5 wt% to about 27 wt% salt.

28. An algal aquaculture system for growing a microalgae of interest, the system comprising: an algal aquaculture pond containing an algal aquaculture medium, the microalgae of interest, and at least one species of undesired microorganism, a first apparatus comprising a first pressurization device configured to increase the pressure of an initial portion of the algal aquaculture medium from a first pressure to a second pressure toproduce a pressurized algal aquaculture medium at the second pressure; and a first depressurization device configured to decrease the pressure of the pressurized algal aquaculture medium from the second pressure to a third pressure to produce a depressurized algal aquaculture medium at the third pressure; wherein the depressurized algal aquaculture medium contains a decreased quantity of the undesired microorganism relative to the initial portion of the algal aquaculture medium.

29. The algal aquaculture system of claim 28, further comprising at least one of a preconcentration unit, an algal conditioning unit, an algal harvesting unit, and an algal aquaculture medium polishing zone.

30. The algal aquaculture system of claim 28 or 29, wherein the first apparatus is configured to receive the initial portion of the algal aquaculture medium from one of the algal aquaculture pond, the pre-concentration unit, the algal conditioning unit, the algal harvesting unit, and the algal aquaculture medium polishing zone, or wherein the first apparatus is in fluid communication with one or more selected from the group consisting of the algal aquaculture pond, the pre-concentration unit, the algal conditioning unit, the algal harvesting unit, and the algal aquaculture medium polishing zone, and any combination thereof.

31. The algal aquaculture system of any one of claims 28-30, wherein the depressurized algal aquaculture medium is returned to one of the algal aquaculture pond, the pre-concentration unit, the algal conditioning unit, the algal harvesting unit, and the algal aquaculture medium polishing zone.

32. The algal aquaculture system of any one of claims 28-31, further comprising a second apparatus having a second pressurization device configured to increase the pressure of asecond portion of the algal aquaculture medium from an initial pressure to an increased pressure to produce a second pressurized algal aquaculture medium; and a second depressurization device configured to decrease the pressure of the second pressurized algal aquaculture medium from the increased pressure to a decreased pressure to produce a second depressurized algal aquaculture medium; wherein the second depressurized algal aquaculture medium contains a decreased quantity of the undesired microorganism relative to the second portion of the algal aquaculture medium.

33. The algal aquaculture system of claim 32, wherein the second apparatus is configured to receive the second portion of the algal aquaculture medium from one of the algal aquaculture pond, the pre-concentration operation, the algal conditioning operation, the algal harvesting operation, and the algal aquaculture medium polishing zone.

34. The algal aquaculture system of any one of claims 32 and 33, wherein the second depressurized algal aquaculture medium is returned to one of the algal aquaculture pond, the preconcentration unit, the algal conditioning unit, the algal harvesting unit, and the algal aquaculture medium polishing zone, or wherein the second apparatus is in fluid communication with one or more selected from the group consisting of the algal aquaculture pond, the pre-concentration unit, the algal conditioning unit, the algal harvesting unit, the algal aquaculture medium polishing zone, and any combination thereof.

35. The algal aquaculture system of any one of claims 28-34, wherein the microalgae of interest has a cell membrane but not a cell wall.

36. The algal aquaculture system of any one of claims 28-35, wherein the microalgae of interest comprises at least one member selected from the group consisting of Dunaliella sp.,Dunaliella salina, Dunaliella kone, and Dunaliella bardawil.

37. The algal aquaculture system of any one of claims 28-36, wherein the undesired microorganisms comprise at least one member selected from the group consisting of Heteramoeba sp., Euplotes sp., Blepharisma halophila, Cladotricha sigmoidea, Nassula sp., Parartemia sp., Fabrea salina, and Artemia salina.

38. The algal aquaculture system of any one of claims 28-37, wherein the undesired microorganisms comprise at least one member selected from the group consisting of ciliates, protozoa, amoeba, rotifers, aquatic crustaceans, and Artemia salina.

39. The algal aquaculture system of any one of claims 28-38, wherein the first apparatus is configured to decrease the pressure within one of less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

40. The algal aquaculture system of any one of claims 32-34, wherein the second apparatus is configured to decrease the pressure within one of less than 1 hour, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

41. The algal aquaculture system of any one of claims 28-40, wherein at least one of the depressurized algal aquaculture medium and the second depressurized algal aquaculture medium is substantially free of the undesired microorganisms.

42. The algal aquaculture system of any one of claims 28-41, wherein at least one of the depressurized algal aquaculture medium and the second depressurized algal aquaculture medium contains less than 50% of an initial quantity of the undesired microorganisms, less than 45% of the initial quantity, less than 40% of the initial quantity, less than 35% of the initial quantity, less than 30% of the initial quantity, less than 25% of the initial quantity, less than 20% of the initial quantity, less than 15% of the initial quantity, less than 10% of the initial quantity, less than 5% of the initial quantity, less than 3% of the initial quantity, less than 1% of the initial quantity, and less than 0.5% of the initial quantity.

43. The algal aquaculture system of any one of claims 28-42, wherein the first apparatus comprises at least one of a pump, a piston, a compressor, and an injector to increase the pressure of the initial portion algal aquaculture medium.

44. The algal aquaculture system of any one of claims 32-34 and 40, wherein the second apparatus comprises at least one of a pump, a piston, a compressor, and an injector to increase the pressure of the second portion algal aquaculture medium.

45. The algal aquaculture system of any one of claims 28-44, wherein the first apparatus comprises at least one of a nozzle, a valve, an orifice, and an expansion to decrease the pressure of the pressurized algal aquaculture medium.

46. The algal aquaculture system of any one of claims 32-34, 40, and 44, wherein the second apparatus comprises at least one of a nozzle, a valve, an orifice, and an expansion to decrease the pressure of the second pressurized algal aquaculture medium.

47. The algal aquaculture system of any one of claims 28-46 wherein the undesired microorganisms comprise at least one member selected from the group consisting of: Arthrospira sp., Arthrospira (Spirulina) obliquus, Arthrospira (Spirulina) platensis, Asteromonas sp.,Asteromonas gracilis, Cryptomonas sp., Dunaliella sp., Dunaliella bioculata, Dunaliella granulata, Dunaliella maritima, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella polymorpha, Dunaliella primolecta, Dunaliella pseudosalina, Dunaliella quartolecta, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Eremosphaera sp., Euglena sp., Euglena gracilis, Euglena viridis, Halospirulina sp., Isochrysis sp., Isochrysis galbana, Isochrysis litoralis, Isochrysis maritima, Lepocinclis sp., Lepocinclis acus, Ochromonas sp., Pavlova sp., Pavlova lutheri, Pavlova salina, and Pavlova viridis.

48. The algal aquaculture system of any one of claims 28-47, wherein the increase from the first pressure to the second pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.3 bar), or at least about 10 psi (0.7 bar), or at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar).

49. The algal aquaculture system of any one of claims 28-48, wherein the decrease from the second pressure to the third pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.3 bar), or at least about 10 psi (0.7 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar).

50. The algal aquaculture system of any one of claims 28-49, wherein the difference between the second pressure and the third pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.3 bar), or at least about 10 psi (0.7 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 60 psi (4.1 bar), or at least about 70 psi (4.8 bar), or at least about 80 psi (5.5 bar), or at least about 90 psi (6.2 bar), or at least about 100 psi (6.9 bar), or at least about 110 psi (7.6 bar), or at least about 120 psi (8.3 bar), or at least about 130 psi (9.0 bar), or at least about 140 psi (9.7 bar), or atleast about 150 psi (10.3 bar), or at least about 160 psi (11.0 bar), or at least about 170 psi (11.7 bar), or at least about 180 psi (12.4 bar), or at least about 190, or at least about 200 psi (13.8 bar).

51. The algal aquaculture system of any one of claims32-34, 40, 44 and 46, wherein the increase from the initial pressure to the increased pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.3 bar), or at least about 10 psi (0.7 bar), or at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar).

52. The algal aquaculture system of any one of claims 32-34, 40, 44, 46, and 51, wherein the decrease from the increased pressure to the decreased pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.3 bar), or at least about 10 psi (0.7 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 100 psi (6.9 bar), or at least about 150 psi (10.3 bar), or at least about 200 psi (13.8 bar).

53. The algal aquaculture system of any one of claims 32-34, 40, 44, 46, 51, and 52, wherein the difference between the increased pressure and the decreased pressure is at least about 3 psi (0.2 bar), or at least about 5 psi (0.3 bar), or at least about 10 psi (0.7 bar), or at least about 15 psi (1.0 bar), or at least about 20 psi (1.4 bar), or at least about 30 psi (2.1 bar), or at least about 50 psi (3.4 bar), or at least about 60 psi (4.1 bar), or at least about 70 psi (4.8 bar), or at least about 80 psi (5.5 bar), or at least about 90 psi (6.2 bar), or at least about 100 psi (6.9 bar), or at least about 110 psi (7.6 bar), or at least about 120 psi (8.3 bar), or at least about 130 psi (9.0 bar), or at least about 140 psi (9.7 bar), or at least about 150 psi (10.3 bar), or at least about 160 psi (11.0 bar), or at least about 170 psi (11.7 bar), or at least about 180 psi (12.4 bar), or at least about 190, or at least about 200 psi (13.8 bar).

54. The algal aquaculture system of any one of claims28-53, wherein the first apparatus increases the first pressure to the second pressure occurs in one of less than 1 hour, less than 5 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

55. The algal aquaculture system of any one of claims28-54, wherein the first apparatus decreases the second pressure to the third pressure occurs in one of less than 1 hour, less than 5 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than one second.

56. The algal aquaculture system of any one of claims32-34, 40, 44, 46, and 51-53, wherein the second apparatus increases the initial pressure to the increased pressure occurs in one of less than 1 hour, less than 5 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

57. The algal aquaculture system of any one of claims 32-34, 40, 44, 46, 51-53, and 56, wherein the second apparatus decreases the increased pressure to the decreased pressure occurs in one of less than 1 hour, less than 5 minutes, less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, and less than 1 second.

58. The algal aquaculture system of any one of claims 28-57, wherein the salinity of the algal aquaculture medium is greater than about 5 wt% or greater than about 15 wt%.

59. The algal aquaculture system of any one of claims 28-58, wherein the first apparatus is configured to receive a flow rate of the algal aquaculture medium greater than about 1 m3 / hour, greater than about 5 m3 / h, greater than about 15 m3 / h, greater than about 30 m3 / h, greater than about 50 m3 / h, greater than about 100 m3 / h, or greater than about 200 m3 / h.

60. The algal aquaculture system of any one of claims 28-59, wherein a ratio of thevapor pressure of freshwater to the vapor pressure of algal aquaculture medium is 1.05 - 1.39, optionally at an ambient temperature of 5 - 45°C, such as at 25°C.

61. Use of a first apparatus comprising:(i) a first pressurization device configured to increase the pressure of an initial portion of an algal aquaculture medium from a first pressure to a second pressure to produce a pressurized algal aquaculture medium at the second pressure; and(ii) a first depressurization device configured to decrease the pressure of the pressurized algal aquaculture medium from the second pressure to a third pressure to produce a depressurized algal aquaculture medium at the third pressure; for eradicating undesired microorganisms in an algal aquaculture medium in an algal aquaculture system for growing a microalga of interest.

62. The use of claim 61, wherein the algal aquaculture system is according to anyone of claims 28-60.

63. The use of claim 61 or claim 62, wherein the use is for eradicating undesired microorganisms according to any one of claims 1-27.

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