System for producing rotifer and method for producing rotifer

Abstract

The present disclosure provides a system for producing rotifers, the system comprising a bacterial culture tank and a rotifer culture tank, wherein bacteria are cultured in the bacterial culture tank using an algal culture supernatant as feed, the cultured bacteria are supplied to the rotifer culture tank, and rotifers are cultured in the rotifer culture tank using the bacteria as feed.

Description

System for Producing Rotifers and Method for Producing Rotifers

[0001] The present disclosure relates to a system for producing rotifers and a method for producing rotifers.

[0002] In aquaculture, sustainable feed procurement is required. Microalgae and rotifers are widely used as aquaculture feeds (Non-Patent Documents 1 and 2). Microalgae can grow independently through photosynthesis, while culturing rotifers requires about five times their body weight in food, so a stable supply of rotifer feed is an issue (Non-Patent Document 3).

[0003] Conventionally, organisms such as Nannochloropsis, freshwater Chlorella, and baker's yeast have been used as feed for rotifers (Non-Patent Document 3). Since Nannochloropsis is a photosynthetic organism, light is required for growth, and it is easily affected by the weather. Also, its nutritional value easily changes depending on the culture conditions, and there are problems with the stability of quality (Non-Patent Document 4). Freshwater Chlorella has a high nutritional value and stable quality, but there is a problem of high cost (Non-Patent Document 4). Furthermore, baker's yeast and freshwater Chlorella are not marine organisms, so they die within a short period in a rotifer culture tank, and there is also a problem that the period effective as feed is limited (Non-Patent Document 3).

[0004] From such a background, there is a demand for a feed supply system that can be stably used in seawater and is excellent in terms of cost as feed for rotifers.

[0005] Fukada, Haruhisa et al., Effects of complete replacement of fish oil with plant oil mixtures and algal meal on growth performance and fatty acid composition in juvenile yellowtail Seriola quinqueradiata, Journal of the Japanese Society of Fisheries Science, 87(4), p.314, 2021. Lubzens, E et al., Rotifers as food in aquaculture, Hydrobiologia, Vol. 186, pp. 387-400, 1989. Fisheries Research Agency, Aquaculture Center, Rotifer Course, http: / / ncse.fra.affrc.go.jp / 15kouza / index.html. Okauchi, Masanori et al., Differences in nutritional value of Nannochloropsis oculata in different growth phases, Journal of the Japanese Society of Fisheries Science, 56(8), pp. 1293-1298. 1990.

[0006] This disclosure is made to solve the problems described above and aims to provide a system for producing rotifers and a method for producing rotifers.

[0007] One aspect of the present disclosure is a system for producing rotifers, comprising a bacterial culture tank and a rotifer culture tank, wherein bacteria are cultured in the bacterial culture tank using the culture supernatant of algae as feed, the cultured bacteria are supplied to the rotifer culture tank, and rotifers are cultured in the rotifer culture tank using the bacteria as feed. Another aspect of the present disclosure is a method for producing rotifers, comprising the steps of: cultivating bacteria using the culture supernatant of algae as feed; and cultivating rotifers using the bacteria as feed.

[0008] This disclosure provides a system for producing rotifers and a method for producing rotifers.

[0009] Figure 1 is an overall configuration diagram showing one embodiment of the system for producing rotifers according to the present disclosure. Figure 2 is a flowchart showing one embodiment of the method for producing rotifers according to the present disclosure.

[0010] Non-limiting embodiments of this disclosure will be described below with reference to the drawings. This disclosure is not limited to the embodiments described below.

[0011] <System for producing rotifers> One embodiment of the present disclosure is a system for producing rotifers, comprising a bacterial culture tank and a rotifer culture tank, wherein bacteria are cultured in the bacterial culture tank using the culture supernatant of algae as feed, the cultured bacteria are supplied to the rotifer culture tank, and rotifers are cultured in the rotifer culture tank using the bacteria as feed. In the system of the embodiment, the bacteria may be marine bacteria. The system of the embodiment may further comprise an algae culture tank, in which algae are cultured, and the culture supernatant of the algae can be obtained from the culture of the algae cultured in the algae culture tank. The rotifer production system of the embodiment may further include a centrifuge for solid-liquid separation of a bacterial culture cultured in a bacterial culture tank and / or a centrifuge for solid-liquid separation of an algal culture cultured in an algal culture tank, wherein the bacterial pellets obtained by the centrifuge for solid-liquid separation of the bacterial culture are supplied to the rotifer culture tank, and the culture supernatant of the algae obtained by the centrifuge for solid-liquid separation of the algal culture is supplied to the bacterial culture tank.

[0012] Figure 1 is an overall diagram showing an example of a system for producing rotifers according to an embodiment. The system for producing rotifers shown in the figure includes a bacterial culture tank 1, a rotifer culture tank 2, an algae culture tank 3, a centrifuge 4, and a centrifuge 5.

[0013] The bacterial culture tank 1 is a culture tank in which bacteria are cultured using the culture supernatant of algae as food. The bacteria cultured in the bacterial culture tank 1 are not particularly limited as long as they can be used as food for rotifers, and examples include bacteria of the genera Pseudomonas or Acinetobacter. The bacterial culture tank 1 is designed to be able to culture bacteria suitable as food for rotifers, and it is preferable that it is equipped with a control mechanism to achieve culture conditions suitable for the bacteria, including salt concentration, pH, temperature, and dissolved oxygen concentration. For this reason, it is preferable that the bacterial culture tank 1 is equipped with sensors to monitor one or more of the salt concentration, pH, temperature, and dissolved oxygen concentration, and is equipped with one or more of the following to control these parameters: a device for adding water, salt, pH adjusters, etc., a temperature control device, and a stirring device or an aeration device. It is preferable that the bacterial culture tank 1 is capable of sterile culture of bacteria that serve as food for rotifers. For this reason, it is preferable that the bacterial culture tank 1 is autoclavable. The bacterial culture tank 1 may be equipped with a liquid supply section or a liquid drain section as needed.

[0014] The bacteria cultured in bacterial culture tank 1 may be marine bacteria. Examples of such bacteria include marine Pseudomonas (marine Pseudomonas) or marine Acinetobacter (marine Acinetobacter). When culturing marine bacteria, bacterial culture tank 1 is preferably a culture tank that can handle high salinity, and is preferably at least partially composed of stainless steel, titanium, or other corrosion-resistant materials. Furthermore, when culturing marine bacteria, it is preferable that the culture tank has a control system that can maintain culture conditions including salinity, pH, and temperature suitable for culturing marine bacteria.

[0015] The bacterial culture tank 1 may be equipped with a sampling port for removing bacterial cultures in order to provide a stable supply of bacteria to the rotifer culture tank 2 or the centrifuge 4. The sampling port is preferably configured to allow aseptic removal of bacterial cultures and is preferably equipped with an aseptic valve to prevent contamination. The sampling port may also be configured to allow removal of bacterial cultures while stirring the contents of the bacterial culture tank 1 in order to remove a uniform bacterial culture. Alternatively, the sampling port may be configured to allow removal of bacterial cultures without stirring the contents of the bacterial culture tank 1 so that bacterial flocs formed during cultivation can be recovered.

[0016] The bacterial culture tank 1 may include a transfer system, including a supply pipe and a pump, for continuously supplying bacterial culture to the rotifer culture tank 2 or the centrifuge 4. It is preferable that the supply path for continuous supply of bacterial culture be kept aseptic. The supply system may also include a control system for controlling the flow rate of the transferred bacterial culture to an appropriate level.

[0017] The culture supernatant of algae used as food for bacterial culture in bacterial culture tank 1 is the supernatant obtained by removing the solid components, including cells, or most of them, from the algal culture by solid-liquid separation. The algal culture supernatant may be used after sterilization, including autoclaving or filtration, as necessary. The algae contained in the algal culture are not limited as long as their culture supernatant can be used for bacterial culture. The algae contained in the algal culture may be non-sessile algae. The algae contained in the algal culture may be, for example, unicellular algae or microalgae. As for the algae contained in the algal culture, any of the following classifications may be used: Aurantiochytrium, Chlamydomonas, Chlorella, red algae Schizophyllum, Spirulina, Botryococcus, Euglena, haptophytes, Prasinophytes, green algae, brown algae, red algae, cyanobacteria, diatoms, yellow-green algae, golden algae, dinoflagellates, and seaweed. It is preferable that the algae contained in the algal culture are marine algae. More specific examples of algae include those of the genera Chaetoceros, Isochrysis, Pavlova, Pyramimonas, and Tisochrysis, with a preference for algae of the genus Chaetoceros, such as Chaetoceros gracilis or Chaetoceros carcitrans. Algal culture can be carried out using culture media for algae known to those skilled in the art, seawater, diluted seawater, artificial seawater, mixtures thereof, or solutions having a partially common composition. The culture media for algae can be any aqueous solution in which natural algae can grow without particular limitations, or an aqueous culture medium for algae with a specified composition can be used. The culture medium may be an algal culture medium known to those skilled in the art, and may, for example, be a medium containing nutrients, a carbon source, rare metals, etc. Specific examples of the aqueous culture medium include IMK medium, SWM-3 medium, modified SWM-3 (mSWM-3) medium, modified media of these media, and media obtained by mixing these media or modified media.IMK medium contains 200mg / L NaNO3, 1.4mg / L Na2HPO4, 5mg / L K2HPO4, 2.68mg / L NH4Cl, 5.2mg / L Fe-EDTA, 0.332mg / L Mn-EDTA, 37.2mg / L Na2-EDTA, 0.023mg / L ZnSO4・7H2O, 0.014mg / L CoSO4・7H2O, 0.0073mg / L Na2MoO4・2H2O, 0.0025mg / L CuSO4・5H2O, 0.0017mg / L H2SeO3, 0.2mg / L thiamin-HCl, 0.0015mg / L biotin, 0.0015mg / L vitamin B12 0.18mg / L A medium consisting of MnCl2・4H2O and the balance seawater. In particular, when culturing diatoms, 0.2–1 mM Na2SiO3 may be added to the IMK medium in addition to the above components. mSWM-3 medium contained 17 mg NaNO3, 1.56 mg NaH2PO4・2H2O, 5.68 mg Na2SiO3・9H2O, 1.12 mg Na2EDTA・2H2O, 0.084 mg Fe-EDTA, 0.0346 μg Na2SeO3, 1 ml P-1 metal solution (618.3 mg H3BO4, 69.25 mg MnCl2・4H2O, 5.45 mg ZnCl2, 238 μg CoCl2・6H2O, 100ml distilled water), 0.2 μg vitamin B12, 1 mL Vitamin mixed solution S3 (5 mg thiamine HCl, 1 mg nicotinic acid, 1 mg calcium pantothenate, 0.1 mg p-aminobenzoic acid, 0.01 mg biotin, 50 mg inositol, 0.02 mg folic acid, 30 mg thymine, 100 mL The culture medium (pH 7.7-7.8) is prepared by mixing distilled water, 50 mg Tris (hydroxymethyl) aminomethane, and 98 mL of seawater. The algal culture may be grown under natural light and / or artificial light irradiation. The algal culture is carried out in air or CO2. ２This may be accompanied by aeration and / or stirring. The culture supernatant of algae used for bacterial culture may be used for bacterial culture without the addition of other components, or it may be used with additional nutrients that the bacteria can utilize added. In this disclosure, the expression "used as feed" may mean using only the culture supernatant of algae or bacteria as a nutrient source for the cultured organism, or using them as a nutrient source for the cultured organism with additional nutrients added.

[0018] Rotifer culture tank 2 is a culture tank to which bacteria cultured in bacterial culture tank 1 are supplied, and rotifers are cultured using these bacteria as food. The rotifers in this disclosure are not limited to any rotifers that can be used as food for fish. Rotifers may be animals classified as belonging to the order Ploima, family Brachionidae, genus Brachionus, or Brachionus plicatilis sp. complex. More specifically, the rotifers are preferably S-type rotifers, SS-type rotifers (Brachionus rotundiformis), or L-type rotifers (Brachionus plicatilis).

[0019] Rotifers can be cultured in the rotifer culture tank 2 using a culture medium such as seawater, artificial seawater, a mixture of these with freshwater, or an artificial culture solution suitable for rotifer culture. The rotifer culture tank 2 may be a rotifer culture tank that supports any of the following culture methods: batch type, thinning type, or continuous culture.

[0020] In batch culture, rotifers are inoculated into a culture medium in a container and cultured for a certain period. Once the rotifers have grown to a stationary stage, for example, the culture is terminated and the target rotifers are collected from the culture medium. Subsequently, a portion of the culture medium can be subcultured into a new sterile culture medium to start the next batch culture.

[0021] In thinning culture, rotifers are inoculated into a culture medium and allowed to multiply. When the rotifers reach a certain density, a portion of the culture is removed, and a replacement amount of fresh culture medium (for example, the same amount or volume as the removed portion) is added. By repeating this process, long-term cultivation is possible.

[0022] In continuous culture, rotifers are inoculated into a culture medium, and after they enter the logarithmic growth phase, fresh culture medium is continuously supplied while simultaneously continuously withdrawing the same amount of culture solution. This maintains the rotifers in a steady growth state, enabling stable production over a long period.

[0023] The rotifer culture tank 2 may be equipped with an environmental control unit for controlling environmental conditions such as temperature, a liquid supply unit, a drainage unit, a stirring device or an aeration device, etc.

[0024] The supply of cultured bacteria from the bacterial culture tank 1 to the rotifer culture tank 2 can be carried out by a method corresponding to the rotifer culture method. In the subculture (batch) and thinning methods, appropriate amounts of bacterial culture, bacterial pellets, resuspension of bacterial pellets, or bacterial slurry can be added at the time of subculture and rotifer harvesting, respectively. These bacterial-containing feeds can be supplied in small amounts periodically using a measuring cup or quantitative dispensing device. At this time, the pellets, resuspension, or slurry can be uniformly dispersed in the rotifer culture tank by using a water flow or stirring device. In continuous culture, bacteria can be supplied by a supply system including a supply pipe and a pump. It is preferable that the transfer of bacteria to the rotifer culture tank 2 by the supply system be carried out aseptically.

[0025] The algae culture tank 3 is a culture tank in which algae are cultured and the culture supernatant of the cultured algae is obtained from the cultured algae. The cultivation of algae in the algae culture tank 3 may be governed by the description in this specification of "Algae culture supernatant used for bacterial culture in bacterial culture tank 1". The algae culture tank 3 is preferably a culture tank that can withstand high salinity and is preferably at least partially constructed of stainless steel, titanium, or other corrosion-resistant material. The algae culture tank 3 may have different shapes, including tank-shaped, well-shaped, or tubular, depending on the type of algae to be cultivated and the individual purpose of implementation, and may be equipped with a general configuration for culturing algae, such as an environmental control unit for controlling environmental conditions such as temperature, a liquid supply unit, a drainage unit, a light irradiation device, and a stirring device or aeration device, as needed.

[0026] The centrifugal separator 4 is a centrifugal separator for separating the solid-liquid mixture of the bacterial culture cultured in the bacterial culture tank 1. The bacterial pellets or bacterial slurry obtained in the centrifugal separator 4 are supplied to the rotifer culture tank 2.

[0027] The centrifuge 4 is preferably at least partially constructed of a corrosion-resistant material such as stainless steel or titanium. The centrifuge 4 may be capable of using a large-capacity centrifugal bottle and a corresponding rotor. In the centrifuge 4, the bacterial culture cultured in the bacterial culture tank 1 is separated into solid and liquid components, and the solid component containing bacteria (bacterial pellets or bacterial slurry) is supplied to the rotifer culture tank. Before being supplied to the rotifer culture tank, the bacterial pellets and bacterial slurry may be resuspended, for example, in seawater or a mixture of seawater and freshwater. The centrifuge 4 may be a continuous centrifuge, in which case the centrifuge 4 may receive the bacterial culture from the bacterial culture tank 1 through a sterile supply pipe.

[0028] The centrifuge 5 is a centrifuge for separating the solid-liquid mixture of algae cultured in the algae culture tank 3. The culture supernatant obtained by the centrifuge 5 is a liquid that contains almost no algal cells or other fine particles, and by supplying this to the bacterial culture tank 1, efficient bacterial growth is possible. On the other hand, the algal cells obtained by the centrifuge 5 can be used for fish feed or substance production, etc. The centrifuge 5 may be capable of using a large-capacity centrifugal bottle and a corresponding rotor. The algal culture supernatant obtained by the centrifuge 5 is supplied to the bacterial culture tank 1. It is preferable that the centrifuge 5 is at least partially constructed of a corrosion-resistant material such as stainless steel or titanium. The centrifuge 5 may be a continuous centrifuge, in which case the centrifuge 5 may receive the algal culture from the algae culture tank 3 through a sterile supply pipe. In the embodiment, the centrifuge 4 may use the same drive unit and / or rotor as the centrifuge 5, or a different drive unit, rotor, or a combination thereof may be used.

[0029] <Method for Producing Rotifers> Another embodiment of the present disclosure is a method for producing rotifers, comprising the steps of culturing bacteria using the culture supernatant of algae as feed, and culturing rotifers using the bacteria as feed. In the rotifer production method of this embodiment, the bacteria may be marine bacteria. The rotifer production method of this embodiment may include the steps of culturing algae and obtaining the culture supernatant by solid-liquid separation of the algal culture. The rotifer production method of this embodiment may include the step of solid-liquid separation of the bacteria cultured in the bacterial culture step, in which case the bacterial pellets or bacterial slurry obtained in the solid-liquid separation step may be used as feed in the rotifer culture step. The elements of this embodiment (algae, culture supernatant, bacteria, rotifers, marine bacteria, etc.) may be described in the section <System for Producing Rotifers>.

[0030] An example of a method for producing rotifers according to the embodiment will be explained with reference to the flowchart in Figure 2.

[0031] In step S1, algae are cultured in the algae culture tank 3. This algae culture is performed to obtain the culture supernatant, which serves as food for bacteria. The type of algae cultured is, for example, Chaetoceros gracilis or Chaetoceros carcitrans. The algae culture is carried out under appropriate conditions using a culture medium for algae, which contains seawater, artificial seawater, or a mixture of these and freshwater.

[0032] In step S2, the algal culture is separated into solid and liquid using a centrifuge 5 to separate the algal cells from the culture supernatant. The algal cells obtained in this process are collected as pellets and used as feed for fish and shellfish.

[0033] In step S3, the culture supernatant of the algae obtained in the previous step is supplied to the bacterial culture tank 1. The culture supernatant is used as a nutrient source for the bacteria, and the bacteria are cultured.

[0034] In step S4, the starter culture is added to the bacterial culture tank 1. As the starter culture, a marine bacterium of the genus Pseudomonas or Acinetobacter that can be used as food for rotifers may be used.

[0035] In step S5, bacteria are cultured in bacterial culture tank 1. During this process, culture conditions such as temperature, pH, salinity, shaking speed, and aeration amount are controlled to ensure optimal bacterial growth.

[0036] In step S6, the cultured bacteria are separated into solid and liquid using the centrifuge 4, and the bacterial cells are recovered.

[0037] In step S7, the recovered bacterial pellets or bacterial slurry are supplied to the rotifer culture tank 2.

[0038] In step S8, rotifers are cultured in the rotifer culture tank 2. Brachionus species, specifically S-type or L-type rotifers, can be used. The rotifers are cultured under appropriate conditions using a suitable culture medium containing seawater, artificial seawater, or a mixture of these with freshwater. This allows for efficient production of rotifers for use as fish feed.

[0039] This disclosure includes the following embodiments: [Subject 1] A system for producing rotifers, comprising a bacterial culture tank and a rotifer culture tank, wherein bacteria are cultured in the bacterial culture tank using the culture supernatant of algae as feed, and the cultured bacteria are supplied to the rotifer culture tank, and rotifers are cultured in the rotifer culture tank using the bacteria as feed. [Subject 2] The system according to Subject 1, wherein the bacteria are marine bacteria. [Subject 3] The system according to Subject 1 or 2, further comprising an algae culture tank, wherein algae are cultured in the algae culture tank, and the culture supernatant of algae is obtained from the culture of algae cultured in the algae culture tank. [Clause 4] The system according to any one of Claims 1 to 3, further comprising a centrifuge for solid-liquid separation of a bacterial culture cultured in the bacterial culture tank and / or a centrifuge for solid-liquid separation of an algal culture cultured in the algal culture tank, wherein a bacterial pellet obtained by the centrifuge for solid-liquid separation of the bacterial culture is supplied to the rotifer culture tank, and the culture supernatant of the algae obtained by the centrifuge for solid-liquid separation of the algal culture is supplied to the bacterial culture tank. [Clause 5] A method for producing rotifers, comprising the steps of: culturing bacteria using the culture supernatant of algae as feed; and culturing rotifers using the bacteria as feed. [Clause 6] The method according to Claim 5, wherein the bacteria are marine bacteria. [Clause 7] The method according to Claim 5 or 6, further comprising the steps of: culturing algae; and solid-liquid separation of the algal culture to obtain the culture supernatant. [Clause 8] The method according to any one of claims 5 to 7, comprising a step of solid-liquid separation of the bacteria cultured in the step of culturing the bacteria, wherein the bacterial pellets obtained in the solid-liquid separation step are used as feed in the step of culturing the rotifers.

[0040] While this disclosure has been described with reference to several embodiments described above, this disclosure is not limited to the examples given in these embodiments. Various modifications can be made to the configuration and details of the present invention within the scope of this disclosure.

[0041] 1. Bacterial culture tank 2. Rotifer culture tank 3. Algae culture tank 4. Centrifugal separator 5. Centrifugal separator

Claims

1. A system for producing rotifers, comprising a bacterial culture tank and a rotifer culture tank, wherein bacteria are cultured in the bacterial culture tank using the culture supernatant of algae as feed, the cultured bacteria are supplied to the rotifer culture tank, and rotifers are cultured in the rotifer culture tank using the bacteria as feed.

2. The system according to claim 1, wherein the bacteria are marine bacteria.

3. The system according to claim 1 or 2, further comprising an algae culture tank, wherein algae are cultured in the algae culture tank, and the culture supernatant of the algae is obtained from the culture of the algae cultured in the algae culture tank.

4. A method for producing rotifers, comprising the steps of: culturing bacteria using the culture supernatant of algae as feed; and culturing rotifers using the bacteria as feed.

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