Method for rearing and / or keeping aquatic organisms in a body of water

Abstract

The present invention relates to a method for rearing and / or keeping aquatic organism in a body of water, wherein the method comprises a step for adding carbonic anhydrase polypeptide to the living water.

Description

[0001] Method for rearing and / or keeping aquatic organisms in a body of water

[0002] The invention is related to a method for rearing and / or keeping aquatic organisms in a body of water. The invention relates to a method for reducing the level of CO2 in a body of water comprising aquatic organisms. The invention also relates to a method for increasing among others specific growth rate of each organism, improving welfare of the organisms and / or increasing the biomass in a body of water.

[0003] Background of the invention

[0004] Rearing aquatic organisms in bodies of water, such as cages, tanks or ponds is a well known and large industry. The most common organism is fish, for instance salmonids, and most of the fish is reared in fish cages in the sea. Onshore aquaculture is however continuously growing, and has already exceeded 200,000 tons annually just for Atlantic salmon in onshore tanks.

[0005] The total biomass, or number of animals, allowable in one volume such as a tank or pond onshore is regulated by requirements to the quality of the living water, for instance by setting maximum allowable limits of CO2 and ammonia. These limits are set considering the welfare of the animals, and will be reached before the tank becomes too crowded. By controlling the quality of the living water one may rear more organisms in the same tank at the same time, and thereby improving the economics of plants. The same will apply to tanks on a boat, such as a well-boat, and closed cages or tanks arranged in the sea.

[0006] The compounds of the water mainly influencing the water quality in this regard, are compounds being consumed such as oxygen, and compounds produced by the metabolism of the organisms being reared or other organisms in the water, e.g. bacteria, such as CO2, and ammonia. How much CO2 the organisms produce and how much O2 they consume, are related to the amount of feed, biomass, type and size of the animal, how active the animal is, and to the temperature in the water. As the concentration of CO2 in the living water increases, it becomes harder for the fish to dispose of the CO2 and take up O2, because the concentration difference, and hence the driving force of CO2, between the water and the fish is reduced, which is linked to, and therefore impacts, the uptake of oxygen. Increased CO2 concentration in the fish reduces the ability of the blood to transport oxygen, and affects the fish health.

[0007] The acceptable level or tolerance limits of CO2 of the aquatic organisms in the living water, varies with the type, age and size of the organism, as well as time of exposure. The maximum recommended limit to secure good growth and welfare of Atlantic salmon is 15 mg / L in the living water, however, the limit varies with different species.

[0008] Even at sublethal levels of elevated CO2 in the living water, the growth is affected, and there is a near linear negative correlation between CO2 concentration in the living water and the specific growth rate of Atlantic salmon, in the range of 5-40 mg / L CO2. If the concentration of CO2 in the living water becomes too high, it is toxic to the animals and affects not only the respiration, but also the pH balance, sensory and nervous system negatively, and may result in unconsciousness and death at high levels. It also makes the fish more vulnerable for other qualities of the water, such as low concentration of O2 or high concentration of ammonia which can allow opportunistic pathogens to infect animals causing disease, reduction in health, and economic loss. The CO2 concentration is thus directly affecting the economics of the aquaculture system as well as animal health and welfare in aquaculture systems and aquaria.

[0009] Oxygen may be supplied as needed directly into the tank, whereas the CO2 and ammonia levels are often controlled by replacing part of the water in the tank with water having lower concentrations of CO2 and ammonia. This is typically done by simultaneously adding and removing water to tank, typically adding and removing water from different positions of the tank, but could also be done by first removing some of the water and then replacing it with new water. The water added may be new water such as in flow-through systems and / or it may be recycled water that has been treated such as in Recirculation Aquaculture Systems (RAS). There are known several systems for treating and recycling the water in a tank, and such systems often comprise a degasser for removing CO2, a biofilter for removing ammonia and separators and filters to remove particles. These systems may be arranged to recycle water in a tank onshore, on a boat such as well-boat or on a barge for offshore tanks. Typically, concerning CO2, new water in equilibrium with the atmosphere contains the lowest level, whereas the CO2 level in treated reused water depends on the efficiency of the degasser. The efficiency of the degasser is limited by diminishing efficiency improvements from increasing the height of the degasser and / or the air:water ratio. Increasing the footprint of the degasser is costly.

[0010] The growth rate of the animal and animal welfare are both reduced with increasing CO2 and / or ammonia concentration in the water in the tank. During regular rearing, the limit for CO2 will be reached before the limit for ammonia. If the problems related to CO2 are solved the water replacement necessary to maintain good water quality in the tank can be lowered before the ammonia level gets limiting. Alternatively, the biomass density (number*average weight / water volume) can be increased for the same water replacement rate of the tank yet not reaching the limits of CO2 and ammonia.

[0011] There are several well-known ways to lower the CO2 concentration in the tank. The first is to replace the water in the tank with water having a lower CO2 concentration. However, increasing the water replacement in the tank by increasing the water flow rate would result in higher energy costs for pumping the water and could result in the need for larger equipment such as pumps with higher capacity and changed pipe dimensions. Increased water flow rate of the tank may further create problems with hydraulics in the fish tank with negative impact on the fish health and growth as too high swimming rates stress and exhaust the fish. For RAS, an increase in the water flow rate can cause problems with flooding in the degasser. Another well known way is to increase the pH by adding base or buffer to the tank to shift the equilibrium and move more of the CO2 that is produced into bicarbonate (that is not toxic to the fish). At pH levels relevant for aquatic organisms (pH 6 - 8.5) the CO2 is mainly in the forms of toxic CO2 and harmless bicarbonate. Small shifts in pH make large changes in the relative amounts of CO2 and bicarbonate. The concentration of CO2 in the water is thus dependent both on the gas-liquid balance and on the pH of the water. However, by increasing the pH, the ammonia that is produced becomes more toxic as the equilibrium shifts to more of the toxic NH3 form: NH3 + H+ = NH4+. The upper limit of pH in a typical salmon RAS is normally in the area of 7.7-7.8 to avoid getting more than 20 pg / L NH3-N. Increasing the pH to lower the CO2 concentration will thus just create a new problem. Yet another well known way is to use a device to degas the water directly in the tank by bringing it in contact with air, such as with air diffusors, a surface aerator, a paddle wheel, a water fall, a fountain, or other devices that throw the water into the air above the water body or bring air into the water body that is subsequently leaving the water body enriched with CO2. However, installation of such devices is costly and not very effective for high densities of animals producing a lot of CO2 other places than the surface layers of the water body. Also, the process creates turbulence and bubbles that can have negative effects by stressing and disturbing the organisms.

[0012] It is known, Norwegian patent NO347340 to use carbonic anhydrase enzyme (hereinafter referred to as CA or CA enzyme or polypeptide having carbonic anhydrase activity) in a degasser to remove CO2 from the water being used to rear aquatic organisms. The enzyme is immobilised in a bed, and the water and gas flows through the bed.

[0013] CO2 will naturally be converted to its ionic forms, such as carbonate (CO32) and bicarbonate (HCOs’) in the living water. The gas CO2 is quite soluble in water and more than 99% exists as the dissolved gas and less than 1 % as carbonic acid H2CO3 (Eq. 1 ). The carbonic acid partly dissociates to give H+, HCOs- (Eq. 2) and CO32- (Eq. 3).

[0014] Eq. 1 : CO2 + H2O = H2CO3 (carbonic acid)

[0015] Eq. 2: H2CO3 = H++ HCO3' (bicarbonate)

[0016] Eq. 3: HCO3’ = H++ CO32- (carbonate) The CA enzyme increases the speed of Eq. 1 which is the rate limiting step above, and thereby more bicarbonate can be converted to CO2 in the degasser during the retention time, and as a result more CO2 can be removed in the degasser using the CA enzyme. However, the enzyme gets degenerated over time, and it may be costly and time consuming to replace the bed in the degasser, as well as to immobilize the enzyme to the media bed. Further, precautions must also be taken to ensure that no toxic coating chemicals from the immobilization are getting into the rearing system.

[0017] It is known from WO 2019 / 212359, that the CA enzyme may be immobilized on a gas separation membrane transferring CO2. Gas transferring membranes are however expensive and less suitable for treating large amounts of water.

[0018] US 2015010453 and US 2015231561 describe processes for removal of CO2 from gas by bringing the gas in contact with a liquid and CA enzymes. However, the process equipment and operating conditions for removing CO2 from gas are very different from the processes and conditions for removing CO2 from liquid.

[0019] An object of the invention is to provide a method for rearing and / or keeping aquatic organism in a body of water, without being limited by the water quality, such as the CO2 levels. Another object is to lower the feed conversion ratio (FCR) and increase the specific growth rate (SGR) in order to improve the economics of the plants. Further, it is an object to increase the number of individual organisms and the biomass in the body of water. As always, it is an object to consider and improve the welfare of the reared organisms.

[0020] Another object of the invention is to reduce the need for recycling and / or diluting the living water, and to reduce the size of a degasser in a recycling loop. Yet another object is that it should be possible to carry out the method in existing rearing and / or keeping systems. Yet another object is that the method should provide a stable and reliable water quality not reducing the fish welfare and growth. Description of the Sequences

[0021] SEQ ID NO: 1 is the polynucleotide sequence from Persephonella marina encoding for the carbonic anhydrase DSM 14350.

[0022] SEQ ID NO: 2 is the amino acid sequence of the carbonic anhydrase DSM 14350 SEQ ID NO: 3 is the polynucleotide sequence encoding a carbonic anhydrase.

[0023] SEQ ID NO: 4 is the amino acid sequence of a carbonic anhydrase encoded by SEQ ID NO 3.

[0024] SEQ ID NO: 5 is the amino acid sequence of a polypeptide from bovine erythrocytes having carbonic anhydrase activity.

[0025] SEQ ID NO: 6 is the amino acid sequence of a polypeptide having carbonic anhydrase may be a carbonic anhydrase from Sintef, SCA-11 , as described in WQ201 4090327.

[0026] Summary of the Invention

[0027] The above said objects are solved by a method according to the characterizing part of the independent claims. Further advantageous features are given in the corresponding dependent claims.

[0028] The invention is directed to a method for rearing and / or keeping aquatic organisms, such as aquatic animals in a body of water, wherein a polypeptide having carbonic anhydrase activity is added to the water.

[0029] The invention is further directed to a method for increasing weight gain, specific growth rate, lowering the FOR and / or improving the health of aquatic organisms in a body of water, wherein a polypeptide having carbonic anhydrase activity is added to the water.

[0030] Another aspect of the invention is directed to a method of reducing the level of CO2 in a volume of water, said water comprising CO2-expiring aquatic organism and / or animals, comprising adding a polypeptide having carbonic anhydrase activity to the water, wherein the adding leads to dissolution of the polypeptide in the water. A related aspect is directed to a method for reducing the level of CO2 in a body of water, said water comprising aquatic organisms, comprising adding a polypeptide with carbonic anhydrase activity to the body of water.

[0031] The invention relates to a method for rearing and / or keeping CO2 producing aquatic organism in a body of water, wherein the method comprises a step for adding a polypeptide having carbonic anhydrase activity to the living water.

[0032] An aspect of the invention is directed to the use of a polypeptide having carbonic anhydrase activity in a method a. for rearing and / or keeping aquatic organism in a body of water; b. for reducing the level of CO2 in a body of water, said water comprising CO2- expiring aquatic organisms and / or aquatic animals; and / or c. of increasing the weight gain, increasing the specific growth rate, lowering the FCR, or improving the health of an aquatic organism in a body of water, the method comprising adding said polypeptide having carbonic anhydrase activity to said body of water.

[0033] Detailed Description of the Invention

[0034] Definitions

[0035] By " body of water" it should be understood any suitable natural or artificial aquatic environment. The natural or artificial aquatic environment is typically a closed or substantially closed body of water wherein the volume of water does not substantially change and wherein the water exchange, if any, is relatively low. Typically, the natural or aquatic environment is one where the water exchange or inflow of water, such as through a current, is relatively low. Suitable embodiments of a body of water include a farming tank onshore or offshore, another type of tank; an aquarium; a pond; a pool; a paddy; a lake; a partially or fully enclosed or fenced off portion of an organism’s or animal’s natural habitat, such as a pond, a pool, a paddy, a lake, an estuary, a tarpaulin enclosed body of water, a well boat, a marsh, a lagoon. The methods of the invention can occur in any body of water having an aqueous environment where it is advantageous to the aquatic lifeforms to reduce the levels of CO2. By "rearing water" or "living water" it is herein meant the water surrounding the living aquatic animals or other organisms as they are growing. The term “rearing and / or keeping” should be understood to mean bred or raised and it should be understood to include all life stages of the organism or animal, from hatchery to slaughter. Time periods wherein the organisms are kept in the water but not necessarily growing, including for instance organisms in an aquarium and organisms kept in a tank during transportation, should also be included in "rearing and / or keeping".

[0036] The rearing or living water is the body of water, wherein the cultivation, breeding, raising, production, transport, propagation and / or harvesting of the aquatic organism or animal according to the invention occurs, said body of water being any suitable natural or artificial aquatic environment. The animals may, for example, be reared, bred or raised in a body of water such as a fish cage or tank, and the rearing water will be the water in the cage / tank containing the living aquatic animals. Further, if the water is a part of a system, such as a RAS system or another circulating system, "rearing water" or "living water" would refer to the water flowing in the whole system.

[0037] The rearing or living water may have a temperature in the range of -5 to +42 degrees Celsius, preferably 0 to 35 degrees, most preferably in the range of 5 to 30 degrees. The rearing or living water may have a pH in the range of 3 to 1 1 , preferably 4 to 10 and most preferably in the range of 5 to 9. The salinity of the rearing or living water may be in the range of 0 to 40, more preferably in the range of 0 to 35 ppt.

[0038] The methods of the invention may also comprise known steps necessary for rearing and / or keeping animals, such as feeding, removal of waste, recirculation and / or dilution of water, control of water quality, temperature, light etc. Even steps for handling the fish, such as vaccinating, may be included in the method.

[0039] By the methods according to the invention, CA enzyme will be added to the living water, and thereby the concentration of CO2 in the water surrounding the fish will be at equilibrium with the amount of bicarbonate and carbonate in the water, at all times. The CO2 gas produced by the living animals will thus be converted to nontoxic bicarbonate quickly, and thus the CO2 concentration in the water will increase slower. As the concentration of CO2 is lower, the animals will more easily dispose of CO2, and the fish welfare will increase, the growth will increase and the FCR will be reduced.

[0040] When the water in the body of water, such as cage or tank is a part of a water treatment and / or recycling system, such as a RAS system, the water will be treated to remove CO2, for instance in an aerator or degasser. A part of the water in the body of water is separated from the aquatic organism, treated a.o. to remove CO2 and recycled to the body of water holding the animals. Some of the water may be displaced regularly by new water in order to keep a desired quality of the water. The principles of a recycling and / or RAS system are well known to a skilled person. According to the methods of the invention, CA enzyme will be added to the water, and the enzyme will thus also be in the water during the water treatment, and thereby the conversion of bicarbonate to CO2 will go faster in the aerator / degasser. As CO2 is removed, more carbonate and bicarbonate will be converted to CO2 and removed. The total amount of inorganic carbon (CC / carbonate / bicarbonate) removed will thus increase and the water recycled to the body of water will have a lower concentration of both CO2, carbonate and bicarbonate.

[0041] When the concentration of CO2 in the living water is lower, the specific growth rate (SGR) of the animals will increase and the average feed conversion ratio (FCR) will be reduced. Further, as the total amount of inorganic carbon is reduced, the fish may dispose off more CO2 before the CO2 level being in equilibrium with bicarbonate is so high that the water must be treated. This increases both the fish welfare and the economics of the plant.

[0042] By the methods according to the invention, CA will be in the water through the whole process, and the growth rate of the animals will increase and the FCR will decrease. Further, the number of animals or the biomass in the body of water may be increased, the recycling system may be run slower, and / or the size of the degasser can be reduced, without affecting the growth or the FCR of the animals.

[0043] The CA enzyme may be added directly to the water in a body of water containing the animals, or to a system comprising the water, such as a recycling system. Further, the enzyme may be added to a part of the water, such as the water in a bypass pipe, and then the water may be mixed with the water of the system. Regardless of the addition site, the enzyme will be present in the water in the body of water and through the whole system connected to the body of water.

[0044] The CA enzyme may be added to the living water previous to adding of the animals, allowing the enzyme to disperse into the whole system before the animals are added. The CA enzyme may be added to a part of the living water, cut off from the rest of the living water, such as a part of a RAS system. Once the concentration of the CA enzyme is stabilized, the part may be reconnected with the rest of the system The CA enzyme may be added in a higher concentration when it is not added directly to the water containing the living animals. The CA enzyme may be added while the animals are present in the living water, the maximal concentration should not exceed the tolerance of the animals.

[0045] The CA enzyme may be degenerated, or the activity of the enzyme may be reduced over time, and parts of the water in the system may be replaced. This will lower the concentration of active CA enzyme. The degeneration of the CA enzyme, or the reduction in enzyme activity will depend among others on the origin of the enzyme and the temperature. The CA enzyme may therefore be added to the water intermittently or continuously as long as the animals are in the water.

[0046] The enzyme should be dosed to reach a certain carbonic anhydrase activity level in the body of water, however any addition will give some effect. The optimal CA activity level depends on a number of factors such as the specific conditions in the body of water (e.g. temperature, alkalinity, salinity, etc.), the specific aquatic organisms, (e.g. species, age, size, etc.), the specific systems and their operating parameters, and the specific performance objectives, and may thus vary widely. In order to maintain a certain enzyme activity level, additional enzyme dosing may be required.

[0047] The term “polypeptide having carbonic anhydrase activity”, CA enzyme, carbonic anhydrase or “CA activity” is defined herein as an enzyme of the class EC 4.2.1 .1 and which catalyses the conversion between carbon dioxide and bicarbonate [CO2 + H2O HCO3’ + H+], For purposes of the present invention, one unit of CA activity is defined after Wilbur [1 WAU = (1 / tc)-(1 / tu) x 1000] where WAU is units and tcand tu represent the time in seconds for the catalyzed and uncatalyzed reaction, respectively (Wilbur, 1948, J. Biol. Chem. 176: 147-154).

[0048] Polypeptides are considered to have CA activity if they have at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the CA activity of the polypeptide consisting of the amino acid sequence corresponding to amino acid residues 20 to 243 of SEQ ID NO: 2 or amino acid residues of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. CA activity is determined by a method described in Example 2.

[0049] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0050] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0051] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment).

[0052] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0053] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0054] The carbonic anhydrase

[0055] The methods of the invention are directed to the use of carbonic anhydrase. It is an object of the invention to improve the health of an aquatic organism in a body of water comprising adding a polypeptide having carbonic anhydrase activity.

[0056] Any polypeptide having carbonic anhydrase activity under conditions of the rearing or living water may be used. This means any polypeptide having carbonic anhydrase activity in a temperature range of -5 to +42 degrees Celsius, preferably 0 to 35 degrees, most preferably in the range of 5 to 30 degrees, pH of 3 to 11 , preferably 4 to 10 and most preferably in the range of 5 to 9, and salinity range of 0 to 40, more preferably in the range of 0 to 35 ppt may be used by the methods of the invention. The polypeptide having carbonic anhydrase activity may be any of enzyme class EC 4.2.1 .1 . The carbonic anhydrase may be any suitable for use in aquatic systems such as those originating marine environments. Accordingly, in one embodiment, the polypeptide having carbonic anhydrase activity is a polypeptide of marine origin.

[0057] The carbonic anhydrase activity of the polypeptide may be determined by a number of conventional methods known to the person skilled in the art. In a preferred embodiment, carbonic anhydrase activity is determined by a method described in Example 2.

[0058] In an embodiment, the polypeptide having carbonic anhydrase activity is selected from a polypeptide having at least 50% of the carbonic anhydrase activity as any one of SEQ ID: 2, SEQ ID NO: 4 SEQ ID NO: 5 and SEQ ID NO: 6, such as at least 60%, at least 70%, at least 80% of the carbonic anhydrase activity of SEQ ID: 2, SEQ ID NO: 4 SEQ ID NO: 5 or SEQ ID NO: 6 (as determined by an assay of Example 2). In an embodiment, the polypeptide having carbonic anhydrase activity is selected from a polypeptide having at least 50% of the carbonic anhydrase activity by a polypeptide encoded by a polynucleotide sequence as any one of SEQ ID: 1 and SEQ ID NO: 3, such as at least 60%, at least 70%, at least 80% of the carbonic anhydrase activity of a polypeptide encoded by SEQ ID: 1 and SEQ ID NO: 3 (as determined by an assay of Example 2).

[0059] The carbonic anhydrase to be used in the present invention may originate from marine microbes selected from the group consisting of: B. plakortidis P203, Caminibacter mediatlanticus, Persephonella marina, as well as deep sea and shallow sea metagenomes such as the Logatchev hydrothermal field and wastewater.

[0060] The carbonic anhydrase to be used in the present invention may be derived from or producible by bacteria of the genus Persephonella. Several bacterial strains belonging to the genus Persephonella have been isolated from deep-sea hydrothermal vents. Currently, three species have been identified, namely Persephonella marina, Persephonella hydrogeniphila and Persephonella guaymasensis (Gotz, et al., 2002, International Journal of Systematic and Evolutionary Microbiology, 52, 1349-1359 and Nakagawa, etal., 2003, International Journal of Systematic and Evolutionary Microbiology, 53, 863-869). The carbonic anhydrase may originate from marine microbes selected from the group consisting of Persephonella marina, Persephonella hydrogeniphila and Persephonella guaymasensis.

[0061] Persephonella marina has been subjected to genomic sequencing (EMBL-EBI ID CP001230). The open reading frame identified as SEQ ID NO: 1 is predicted to be translated into SEQ ID NO: 2. The cloning, expression and isolation of the mature carbonic anhydrase from P. marina DSM 14350 are described in WO 2012 / 025577 and confirm that the amino acid sequence gives rise to an enzyme with carbonic anhydrase activity.

[0062] The carbonic anhydrase to be used in the present invention may be derived from, obtainable from, or producible by bacteria strains selected from one of the species Persephonella marina, Persephonella hydrogeniphila or Persephonella guaymasensis, preferably derived from or producible by one of the strains deposited as Persephonella marina DSM 14350, Persephonella hydrogeniphila DSM 15103 or Persephonella guaymasensis DSM 14351 .

[0063] In a further embodiment, the polypeptide having carbonic anhydrase activity may be selected from the group consisting of a) a polypeptide having carbonic anhydrase activity in a water having a temperature in the range of -5 to +42 degrees Celsius, a pH in the range of 3 to 11 , and salinity in the range of 0 to 40. b) a polypeptide derived from, obtainable from or produced by a marine microbe, c) a polypeptide derived from, obtainable from or producible by Persephonella marina DSM 14350; d) a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to amino acid residues 20 to 243 of SEQ ID NO: 2 or amino acid residues of SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6; e) a polypeptide having an amino acid sequence corresponding to amino acid residues 20 to 243 of SEQ ID NO: 2 or amino acid residues of SEQ ID NO: 4, SEQ ID NO: 5 OR SEQ ID NO: 6; f) a fragment of any one of (a) - (e) having carbonic anhydrase activity as determined by a method described in Example 2; and g) a polypeptide having at least 50% of the carbonic anhydrase activity as any a polypeptide encoded by any one of SEQ ID: 1 and SEQ ID NO: 3, such as at least 60%, at least 70%, at least 80% of the carbonic anhydrase activity of a polypeptide encoded by any one of SEQ ID: 1 and SEQ ID NO: 3 (as determined by an assay of Example 2); h) a polypeptide having at least 50% of the carbonic anhydrase activity as any one of SEQ ID: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, such as at least 60%, at least 70%, at least 80% of the carbonic anhydrase activity of SEQ ID: 2, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, (as determined by an assay of Example 2); i) a polypeptide encoded by a nucleic acid sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.

[0064] The polypeptide can be synthetic or derived from other species than Persephonella as long as the polypeptide fall within the stated sequence identity and maintains carbonic anhydrase activity.

[0065] The carbonic anhydrase to be used in the present invention may be a Thermovibrio ammonificans carbonic anhydrase (TACA). TACA is a carbonic anhydrase that catalyzes the interconversion of CO2 and water to bicarbonate and hydrogen ions or vice versa. TACA is obtained or derived from the thermophilic bacteria Thermovibrio ammonificans (TA) (Giovannelli D, Ricci J, Perez- Rodriguez I, Hugler M, O'Brien C, Keddis R, Grosche A, Goodwin L, Bruce D, Davenport KW, Detter C, Han J, Han S, Ivanova N, Land ML, Mikhailova N, Nolan M, Pitluck S, Tapia R, Woyke T, Vetriani C. "Complete genome sequence of Thermovibrio ammonificans HB- 1 (T), a thermophilic, chemolithoautotrophic bacterium isolated from a deep-sea hydrothermal vent" Standards in Genomic Science 2012 7:82-90.). Methods for isolating / obtaining an enzyme from bacteria are known, such as immune- precipitation, ultracentrifugation, or chromatographic methods. The TACA to be used in the present invention may be a polypeptide comprising the sequence as set forth in WO16029316 A1 and may be derived from an expression or cloning vector comprising a nucleotide sequence encoding such carbonic anhydrase, or a transgenic cell comprising such expression or cloning vector.

[0066] The polypeptide having carbonic anhydrase may be a carbonic anhydrase from bovine erythrocytes (such as available from Sigma-Aldrich, Product Number C2624 / CAS Number 9001 -03-0). The polypeptide having carbonic anhydrase may be a carbonic anhydrase from Sintef, SCA-11 , as described in WO2014090327. . The sequences are enclosed herein as SEQ ID NO 5 and 6.

[0067] Water conditions may influence the performance of the carbonic anhydrase. Suitable embodiments of carbonic anhydrases are those having activity in the relevant conditions of a volume of water. The activity level for the conversion of CO2 in pond or aquarium water conditions will differ from than those in carbon capture and storage (CCS) applications, as known to the person skilled in the art, since activity will vary at lower temperatures, in presence of salt, in presence of gasses, in presence of organic material that arises from fish / shrimp production and under conditions where compounds like H2S and NOx may be present.

[0068] The enzyme may be formulated by any conventional method. As stated, according to the invention, carbonic anhydrase is added to the water. The carbonic anhydrase may be in solid or liquid form. The carbonic anhydrase may be formulated as a granule, powder, an aqueous solution or the like. The use of the CA enzyme in the methods of the invention do not require immobilization of the enzyme. In a typical embodiment, the enzyme is added to the volume of water so as to dissolve the enzyme within the volume of water. The invention relates to a method for increasing growth of aquatic organisms in a body of water, wherein carbonic anhydrase enzyme (CA enzyme) is added to the living water. The methods of the invention are directed to rearing and / or keeping including cultivation, breeding, raising, production, propagation, transport and / or harvesting of an aquatic organism or animal in a body of water.

[0069] The methods of the invention can supplement or substitute existing methods of maintaining low levels of CO2 such as replacing the water or by exposing the water to a gas, normally air, such as by using a pump and / or degasser.

[0070] The living organisms

[0071] One aspect of the invention is directed to a method of maintaining low levels of CO2 in a body or volume of water or reducing levels of CO2 in a body or volume of water, said water comprising growing aquatic animals or organisms, said method comprising adding an effective amount of carbonic anhydrase (CA) so as to improve growth conditions. The invention relates to a method for reducing the concentration of CO2 in living water containing living aquatic animals or organisms, wherein carbonic anhydrase enzyme is added to the water.

[0072] Aquatic respiration is the process whereby an aquatic organism exchanges respiratory gases with water, obtaining oxygen from oxygen dissolved in water and excreting carbon dioxide and some other metabolic waste products into the water. An aspect of the invention is a method of improving the health and growth of aquatic organisms which are CO2-expiring organisms, meaning that they are producing CO2. In a typical embodiment, the aquatic organism is an aquatic animal. However, although higher plants typically use carbon dioxide and excrete oxygen during photosynthesis, they also respire and, particularly during darkness, many plants excrete carbon dioxide and require oxygen to maintain normal functions. According to the invention, plants are not considered net C02-producing organisms as they generally consume more CO2 than they produce in a day or in the lifespan of the plant. The aquatic lifeforms, animals or organisms in the methods of the invention may be selected from the group consisting of fish, eels, crustaceans, , and any aquatic animal that relies on gill like organs to facilitate breathing under water. The aquatic animals of the methods of the invention may preferably be selected from the group consisting of fish, echinoderms, molluscs, arthropods, amphibians, aquatic reptiles, aquatic birds, and aquatic mammals, typically selected from the group consisting of fish, echinoderms, molluscs and arthropods.

[0073] Examples of fish include but are not limited to amberjack, Atlantic salmon, arapaima, angelfish, artic char, barb, barramundi, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, flounder, goby, goldfish, gourami, grouper, guapote, guppy, haddock, halibut, java, koi, labeo, lai, loach, mackerel, meagre, milkfish, mojarra, molly fish, monkfish, mudfish, mullet, paco, pacific salmon, pangasius, pearlspot, pejerrey, perch, pike, platy, pompano, roach, salmon, sampa, sauger, sea bass, seabream, seahorse, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, swordfish, tench, tetra, terror, tilapia, trout, tuna, turbot, vendace, walleye whitefish, wolffish and yellowtail kingfish. Examples of arthropods include crustaceans such as but not limited to crabs, lobsters, crayfish, shrimps, and prawns. Examples of echinoderms include starfish, brittle stars, sea urchins, sand dollars, sea cucumbers, and sea lilies or crinoids. Examples of mollusks include, but are not limited to, abalone, mussels, clams, snails, oysters, scallop, cockles, squid, cuttlefish, and octopus.

[0074] The methods of the invention are most preferred comprising aquatic organisms being aquatic animals, and even more preferred fish.

[0075] The use of the carbonic anhydrase

[0076] In an embodiment of the invention, the methods relate to a body of water having a recirculating aquaculture system (RAS), a RAS-like system or a closed loop aquaculture system comprising the addition carbonic anhydrase (CA) to lower the levels of CO2 in the water. In a further embodiment, the methods relate to an aquatic system selected from the group consisting of a green pond system, a Biofloc system, and intensive aquaculture pond culture, an extensive pond culture, a hydroponic aquaculture system, commercial or non-commercial aquaria, and cage-culture, said method comprising adding CA to lower the levels of CO2 in the water so as to maintain or improve the viability, growth, proliferation and / or health of an aquatic organism or animal.

[0077] In one embodiment, the methods of the invention are directed to green pond systems, such as for shrimp and warm water fish. In an embodiment, the methods of the invention are applied to Biofloc systems, such as for shrimp and other fish. In an embodiment, the methods of the invention are directed to an aquaculture pond culture, such as an intensive aquaculture pond culture comprising high stocking densities and optionally supplemented with commercial feeds. Typically, this is applied in ponds being regular to small in size. Alternatively, the methods of the invention may be applied to extensive aquaculture pond culture comprising low stocking density, optionally supplemented with commercial feeds, but also involving the animals to forage on native pond fauna. This can be applied to commercial farms, to large ponds (>1 ha) or small ponds.

[0078] In an alternative embodiment, the methods of the invention are applied to closed loop aquaculture systems, including RAS or RAS-like systems. In an embodiment, the methods of the invention are applied to hydroponic aquaculture systems, to commercial aquaria and / or to cage culture, such as a sea cage for salmon or river cages for fish such as tilapia.

[0079] In one embodiment, the methods of the invention are to reduce the need for chemicals such a buffers and reduce the use of electricity for a degasser.

[0080] The enzyme should be dosed to reach a certain carbonic anhydrase activity level in the body of water. The optimal CA activity level depends on a number of factors, and can vary widely. The enzyme may be added to reach a CA activity level of 20 to 1000 kWAU / L, more preferred 50 to 1 ,000 kWAU / L to achieve the desired performance. A lower enzyme activity level can be applied if lower performance is sufficient. A higher dose may be used if higher performance is required.

[0081] In order to maintain a certain enzyme activity level in the body of water over time, additional enzyme dosing may be required to account for loss of enzyme activity due to replacement of water containing CA activity with new water not containing CA activity, and / or due to possible degradation and / or inactivation of the enzyme in the body of water over time. The level or frequency of replacement of water depends on the type of process used. For flow-through systems, the water replacement may be 1 ,000% per day. For RAS, the water replacement may be 1 to 100% per day. The possible degradation and / or inactivation of the enzyme depends on stability of the enzyme and water quality parameters.

[0082] The polypeptide having carbonic anhydrase activity may be added intermittently, for instance when the total amount of inorganic carbon reaches a set level. After addition of the polypeptide, more bicarbonate will be converted to CO2 during removal of CO2 for instance in a degasser, and the total amount of inorganic carbon will thus be reduced.

[0083] The animals to be reared are preferably fish or eels, more preferred a salmonid, and the body of water may be a farming tank onshore or offshore, a pond, an aquarium, or a tank on a well-boat.

[0084] The invention will in the following be described by way of an experiment in small scale tanks and the accompanying results. The following detailed description does not limit the invention. Instead, the scope of the invention is defined by the appended claims. The following experiment is performed in onshore tanks rearing salmon, however, it should be appreciated that the method may be used in other facilities, and for rearing and / or keeping other animals. The mentioning of different features, structures, or characteristics which the invention may comprise means that the particular feature, structure, or characteristic may be included in at least one embodiment. The different features, structures, or characteristics may be combined in the same or different embodiments, and in any combinations.

[0085] Further embodiments of the invention

[0086] 1 . A method of increasing the weight gain, increasing the specific growth rate, lowering the FCR, or improving the health of an aquatic organism in a body of water, comprising adding a polypeptide having carbonic anhydrase activity to said body of water.

[0087] 2. The method according to embodiment 1 , wherein the adding a polypeptide having carbonic anhydrase activity reduces the levels of CO2 in said body of water.3. A method for reducing the level of CO2 in a body of water, said water comprising aquatic organisms, comprising adding a polypeptide with carbonic anhydrase activity to the body of water.

[0088] 4. The method according to any of embodiments 1 to 3, wherein the aquatic organism is a net C02-producing organism, such as an aquatic organism which does not consume CO2, such as an organism which respires, typically an organism negatively affected by CO2 levels, at any stage of its development.

[0089] 5. The method according to any of embodiments 1 to 4, wherein the aquatic organism is a net C02-producing organism

[0090] 6. The method according to any of embodiments 1 to 4, wherein the aquatic organism is not a consumer of CO2.

[0091] Examples

[0092] Example 1

[0093] Eight RAS units, each with a total system capacity of 2,200L, and a fish tank of 1 ,000L, were used in this experiment. 4 units were treated with a method according to the invention, and a CA enzyme were added, and 4 units were used as a control. All units were filled with brackish water, set at the same setpoints (Table 1 ) and supplied with 200L of active biochips from the same matured biofilter source.

[0094] Biochips is a filter media providing surface for biofilm in the biofilter. A sketch of one of the units is shown in Figure 1 , showing the fish tank 1 , a recycling unit 2 comprising filters (not shown), also referred to as RAS treatment loop, and pipes 3 and 4. Carbonic anhydrase enzyme and lye were added directly to the fish tank and is indicated by arrows 5 and 6. In this specific experiment, a degasser was included in the system, but the ventilation on the degasser was off, but other parts of the treatment loop was aerated (biofilter), , and 5 % of the water volume was replaced daily.

[0095] The systems were allowed to run for 2 weeks to allow the biofilters to stabilize prior to introducing the fish.

[0096] Lye (NaOH) was added to control the pH levels in all units, and carbonic anhydrase enzyme was added to the 4 units in the experiment. The enzyme used in the experiment was having the amino acid sequence SEQ ID NO: 4. A liquid solution of the enzyme was used, which contained an enzyme activity of 2,800 kWAU / mL. The dosing of the enzyme was started the day after fish were weighted and restocked evenly to the eight systems following acclimatization, with an initial dose of 277 ml into each unit after which continuous dosing (27.7ml / 24h) began. The continuous dosing was performed to compensate for replacement of water, and possible degradation and / or inactivation of the enzyme.

[0097] Atlantic salmon (n= 659) were randomly transferred into the 8 RAS units where they remained for a 2-week acclimation period. On day 0 of the experiment all fish (n = 559, avg 518g) were lightly anaesthetized (Finquel 0.7g / 10L) weighed / measured and then randomly returned to the RAS units to reach a density of 45kg / m3in each unit. After three weeks, 20 fish were selected from each tank, lightly anaesthetized (Finquel 0.7g / 10L), weighed / welfare scored and returned to their original units. An additional 10 fish were weighed from each unit to calculate the average weights for each unit.

[0098] The average specific growth rate (SRG) is shown in Figure 2, and average feed conversion ratio (FCR) is shown in Figure 3. The average CO2 level mg / L in the tank is shown in Figure 4.

[0099] As Figure 2 shows, the SGR was increased by 15.5 % in the units given carbonic anhydrase enzyme, meaning that the increase of biomass in the unit was 15.5 % higher when carbonic anhydrase enzyme was added.

[0100] As Figure 3 shows, the FCR was reduced by 15.5 % in the units given carbonic anhydrase enzyme, meaning that the amount of feed needed to increase the fish bodyweight by 1 kg was reduced by 15.5 %.

[0101] As Figure 4 shows the average CO2 level in the units given carbonic anhydrase enzyme was 15% lower than the control tanks not given any enzyme.

[0102] Figure 4 shows the average CO2 level (mg / L) in fish tank along the X-axis and days after the first dosing of CA enzyme along the Y-axis. The CA enzyme was, as said above, added as a full dose of enzyme between day 0 and day 1 , after which 10% of dose was added daily over 24h until the end of the experiment. The black line indicates the average CO2 level of the control tanks (n=4) and the grey line indicates the average CO2 level of the treatment tanks (n=4). The black and grey line indicates linear trendline of control and treatment tanks respectively.

[0103] At day 6, in order to avoid supersaturation of the water with total gas pressure that can be harmful to the fish, and since the degasser ventilation was off, it was necessary to add an oxygen diffusor in the water treatment systems and degass some of the nitrogen gas. This also contributed to some CO2 degassing in all systems, hence the drop in the CO2 level to the next day. At day 9, 5-6 fish were removed from each system to maintain biomass at the set limit of 60 kg / m3, resulting in a lower CO2 production during the next hours / days. At day 21 there was high turbidity due to extra drum filter cleaning, the pH setpoint was increased by 0.1 , resulting in drop of CO2 the next days.

[0104] Each line represents an average of four systems, which varied independently, as expected for biological production systems, so the trends given in the dotted lines are more important than the day-to-day variations. As can be seen form Figure 4, the average CO2 in the fish tanks of the systems added enzyme was in average 15% lower than for control tanks. The CO2 concentration of the fish tank tend to increase more in the control system than in the treatment over the three weeks of the experiment, indicating that the difference may increase over time.

[0105] The water quality was also monitored during the trials, see table 2 below. Turbidit Tota Alk Adde

[0106] Average Solid CaC pH (g

[0107] Treatments.4 96 260

[0108] Control 5.3 108 340

[0109] The table shows that it was 1 1 % lower alkalinity in the treatment units, this is presumably a result of the CA enzyme speeding up the conversion of bicarbonate to CO2 in the RAS treatment loop, increasing efficiency of degassing CO2, and thereby more CO2 is removed. The table further shows 24% lower addition of lye (NaOH) in treatment to maintain the same pH setpoint of 7.2 in treatment and control units. Other water quality was good and similar. Based on the above, it is clear that addition of CA enzyme gives better growth of the animals, reduced feed conversion ratio and reduced need for lye to maintain the same pH setpoint. All these factors will result in better rearing and / or keeping of the animals and improved result.

[0110] Example 2: Detection of Carbonic Anhydrase Activity

[0111] The assay for the detection of carbonic anhydrase was described by Wilbur, 1948, J. Biol. Chem. 176: 147-154. The set up is based on the pH change of the assay mixture due to the formation of bicarbonate from carbon dioxide as given in equation 1 :

[0112] [CO2+ H2O HCO3- + H+],

[0113] The activity assay used in this study was derived from the procedure of Chirica et al., 2001 , Biochim. Biophys. Acta 1544(1 -2): 55-63. A solution containing approximately 60 to 70 mM CO2 was prepared by bubbling CO2 at a flow rate of 100 ml / min into 100 ml distilled water using the tip of a syringe approximately 30 minutes prior to the assay. The CO2 solution was chilled in an icewater-bath at 0-4°C. The enzyme sample according to the present assay contained 60 g active enzyme protein (AEP) per litre with an activity level of 2,800 kWAU / ml.

[0114] To test for the presence of carbonic anhydrase, 2 ml of 25 mM Tris-HCI solution adjusted to pH 8.3 with 25 mM HCI (containing sufficient bromothymol blue to give a distinct and visible blue color) were added to two 13x100 mm test tubes chilled in 4°C water-bath. To one tube, 10 microliters of the enzyme containing solution was added, and an equivalent amount of deionized water was added to the second tube to serve as a control. 2 ml of CO2 solution was added very quickly and smoothly to the bottom of each tube. Simultaneously with the addition of the CO2 solution, a stopwatch was started. The time required for the solution to change from blue to yellow was recorded (transition point of bromothymol blue is pH 6-7.6). The production of hydrogen ions during the CO2 hydration reaction lowers the pH of the solution until the color transition point of the bromothymol blue is reached. The time required for the color change is inversely related to the quantity of carbonic anhydrase present in the sample. The tubes must remain immersed in the ice bath for the duration of the assay for results to be reproducible. Typically, the uncatalyzed reaction (the control) takes 40 to 150 seconds for the color change to occur, whereas the enzyme catalyzed reaction is complete between 5 and 20 seconds, depending upon the amount of enzyme protein in the enzyme solution added and depending on the residual activity after heat treatment. Detecting the color change is somewhat subjective but the error for triple measurements was in the range of 0 to 1 sec difference for the catalyzed reaction. One unit is defined after Wilbur [1 U = (1 / tc)- (1 / tu) x 1000] where U is units and tc and tu represent the time in seconds for the catalyzed and uncatalyzed reaction, respectively (Wilbur, 1948, J. Biol. Chem. 176: 147-154). These units are also termed Wilbur-Anderson units (WAU).

[0115] Assay I for Detection of Carbonic Anhydrase Activity

[0116] Polypeptides with carbonic anhydrase activity were solubilized in assay buffer (0.188 M TAPS, 0.30 M NaCI, 50 mg / L Cresol red, pH 9.0) and 25 pL sample was transferred into a transparent, flat-bottom 96-well assay plate. The substrate solution was prepared by carbonating a cold 0.15 M NaCI solution using a standard household sparkling water maker (SodaStream). To determine carbonic anhydrase activity 200 pL cold substrate solution was auto injected (300 pL / s) into the sample and the change in absorbance at 573 nm was recorded for 30 s using a ClarioStar microtiter plate spectrophotometer.

[0117] To calculate the activity for each sample, absorbance curves were normalized by subtracting the sample’s absorbance at reaction completion (flat line in the end), so that all samples had a corrected absorbance value of 0.00 at reaction completion. Reaction times were determined as the times passed from sample injection until the normalized absorbance value reached 0.15. The average times for three replicates (tsample average and tnegative control average, respectively) were used to calculate the WAU score with the following formula:

[0118] WAU=(1 / t_(sample average) -1 / t_(negative control average) )x1000 Assay for normalized WAU activity of carbonic anhydrases at 0.2 mg / mL protein relative to Persephonella marina carbonic anhydrase

[0119] Purified carbonic anhydrases (in 50 mM HEPES, 0.3 M NaCI, pH 7) were diluted to 0.2 mg / mL with 0.15 M NaCI. To generate a standard curve Persephonella marina carbonic anhydrase (in 50 mM HEPES, 0.3 M NaCI, pH 7) was diluted with 0.15 M NaCI to 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL and 0.025 mg / mL, respectively. 16 pL diluted enzyme sample was combined with 224 pL assay buffer (0.188 M TAPS, 0.30 M NaCI, 50 mg / L Cresol red, pH 9.0) and 25 pL sample was transferred into a transparent, flat-bottom 96-well assay plate. The substrate solution was prepared by carbonating a 0.15 M NaCI solution using a standard household sparkling water maker (SodaStream). To determine carbonic anhydrase activity 200 pL substrate solution was auto-injected (100 pL / s) into the sample and the change in absorbance at 573 nm was recorded for 30 s using a TECAN microtiter plate spectrophotometer. Each sample was measured in triplicate.

[0120] The time in seconds to reach the absorbance of 0.2 (t0.2) was determined for each sample. A calibration curve was constructed by plotting 1 / t0.2 vs the concentration of the corresponding standard. The normalized activity of the samples was calculated using linear regression with the standard curve. One normalized WAU activity unit corresponds to the activity of 1 .0 mg / mL Persephonella marina carbonic anhydrase in this assay.

[0121] Assay II for Detection of Carbonic Anhydrase Activity

[0122] CO2 is bubbled into a reactor cell containing a 1 ,45 M KHCO3 / K2CO3 solution with / without enzyme. The pH and the temperature are both measured during the CO2 addition, using probes. Data for the pH measurements are then converted into a CO2 hydration rate, which is temperature corrected and normalized. A standard curve for the CO2 hydration rate at different known concentrations is established for each enzyme. From the standard curve the enzyme concentration can be calculated from the measured CO2 hydration rate.

[0123] Solution 1 .45M K2CO3 a0.55 (5 L) For Activity measurement procedure 1 . Add 4 L of Dl-water into a 5 L flask. Add a suitable magnet

[0124] 2. Vigorously stir the solution on a magnetic stirring plate

[0125] 3. Add 902 g of K2CO3 and 145 g of KHCO3 into the Dl-water

[0126] 4. Stir until the powder is completely dissolved

[0127] 5. Fill up with Dl-water to exactly 5 L and stir again

[0128] Prior to measurements

[0129] 1 . pH meter needs to be calibrated

[0130] 2. CO2 flow is set to 2L / min

[0131] 3. Stirrer is set to 800 rpm

[0132] Measurement

[0133] 1 . Add 20 ml sample into a reactor cell (glass beaker)

[0134] 2. Add 1 ,45M K2CO3 up to 100 ml in the same reactor cell

[0135] 3. Place reactor cell in the CRAB set-up

[0136] 4. Start stirrer and CO2 flow

[0137] 5. Start the pH and temperature data logging as quickly as possible after the CO2 flow has been initiated

[0138] 6. Monitor the CO2 flow throughout the measurement

[0139] 7. Stop the measurement once pH reached 9.40

[0140] 8. Note down the time to reach pH 9,40 and use a standard curve to determine CA concentration

[0141] By comparing the measured rate of different enzymes, the activity of the enzymes in relation to each other, may be determined by taking into account the slope of their standard curves.

[0142] Example 3 - Test of polypeptides in conditions corresponding living water of aquatic animals

[0143] Two polypeptides having carbonic anhydrase activity was tested under conditions corresponding living water of aquatic animals.

[0144] Thermostated glass reactor with glass diffuser and magnetic stirrer

[0145] Cooling bath (Julabo)

[0146] CO2 Sensor : Oxyguard Dissolved CO2 Analyser: 0-50 mg / L (±0,1 ) dissolved CO2 pH sensor: PH METER PH3310 from WTW

[0147] Gas flow controller: ALICAT 0 to 2000 cm3 (STP) / min

[0148] Water: RAS water Hardingsmolt AS, April 3rd2019

[0149] CO2 water content controlled by bubbling 300 cm3(STP) / min of 15% CO2 in N2 for 1 min. Resulted CO2 (aq) = 30 to 50 mg / L

[0150] CO2 stripping by bubbling air, first 1000 and during recorded experiments 300 cm3(STP) / min. air

[0151] Time, CO2 and pH continuously registered from 15 mg / L until 3 mg / L CO2.

[0152] The polypeptides were

[0153] - a carbonic anhydrase from bovine erythrocytes (available from Sigma-Aldrich, Product Number C2624 / CAS Number 9001 -03-0). The sequence of the polypeptide is enclosed herein as SEQ ID NO 5

[0154] - a carbonic anhydrase as described in WO 2014090327. The sequence of the polypeptide is enclosed herein as SEQ ID NO 6

[0155] The water was RAS water from an aquaculture cage, having

[0156] Temperature: 12,9 (±0,1 ) degrees Celsius pH : 7,2

[0157] Salinity: 25 parts per thousand (ppt)

[0158] The amount of CO2 was reduced 1 ,42 times faster when adding 90 g / m3of the polypeptide, as shown in Figure 5. This clearly shows that polypeptides according to SEQ ID NO 5 and 6.

Claims

Claims1 . Method for rearing and / or keeping aquatic organism in a body of water, wherein the method comprises a step comprising adding a polypeptide having carbonic anhydrase activity to the living water.

2. Method for reducing the level of CO2 in a body of water, said water comprising CO2-expiring aquatic organisms, wherein the method comprises a step for adding a polypeptide with carbonic anhydrase activity to the water.

3. A method of increasing the weight gain, increasing the specific growth rate, lowering the FCR, or improving the health of an aquatic organism in a body of water, comprising adding a polypeptide having carbonic anhydrase activity to said body of water.

4. Method according to any one of claims 1 -3, wherein the water has a temperature in the range of -5 to +42 degrees Celsius, a pH in the range of 3 to 11 , and a salinity in the range of 0 to 40.

5. Method according to any one of claims 1 to 4, wherein at least a part of the water in the body of water is recirculated in a recycling loop, and that the polypeptide is added to the water in the recycling loop.

6. Method according to claim 5, wherein the water is treated to remove CO2 in the recircling loop.

7. Method according to any one of the above claims, wherein the CA polypeptide is added regularly.

8. Method according to any one of the above claims, wherein the CA polypeptide is added intermittently.

9. Method according to any one of the above claims, wherein the aquatic organism a an aquatic animal, such as an aquatic animal selected from the group consisting of fish, echinoderms, molluscs and arthropods, such as a fish.

10. Method according to any one of the above claims, where in the polypeptide is derived from marine microbes.1 1 . Method according to any one of the above claims, wherein the polypeptide is derived from or produced by bacteria of the genus Persephonella, such as polypeptide derived from or produced by the strains deposited as Persephonella marina DSM 14350, Persephonella hydrogeniphila DSM 15103 or Persephonella guaymasensis DSM 14351 .

12. The method according to any one of the preceding claims, wherein the polypeptide having carbonic anhydrase activity may be selected from the group consisting of a) a polypeptide having carbonic anhydrase activity in a water having a temperature in the range of -5 to +42 degrees Celsius, a pH in the range of 3 to 1 1 , and salinity in the range of 0 to 40; b) a polypeptide derived from, obtainable from or produced by a marine microbe; c) a polypeptide derived from, obtainable from or producible by Persephonella marina DSM 14350; d) a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to amino acid residues 20 to 243 of SEQ ID: 2, or amino acid residues of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; e) a polypeptide having an amino acid sequence corresponding to amino acid residues 20 to 243 of SEQ ID NO: 2 or amino acid residues of SEQ ID NO:

4. SEQ ID NO: 5 or SEQ ID NO: 6; f) a fragment of any one of (a) - e) having carbonic anhydrase activity as determined by method (as determined by an assay of example 2);g) a polypeptide having at least 50% of the carbonic anhydrase activity as any a polypeptide encoded by any one of SEQ ID: 1 and SEQ ID NO: 3, such as at least 60%, at least 70%, at least 80% of the carbonic anhydrase activity of a polypeptide encoded by any one of SEQ ID: 1 and SEQ ID NO: 3 (as determined by an assay of Example 2); h) a polypeptide having at least 50% of the carbonic anhydrase activity as any one of SEQ ID: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, such as at least 60%, at least 70%, at least 80% of the carbonic anhydrase activity of SEQ ID: 2, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 (as determined by an assay of Example 2); and i) a polypeptide encoded by a nucleic acid sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO:

3.

13. The method according to any of the preceding claims, wherein the adding a polypeptide having carbonic anhydrase activity reduces the levels of CO2 in said body of water.

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