TWI757003B - Candida palmioleophila sw4’-w6 and method for treating water in culture pond using the same - Google Patents

Abstract

A strain of Candida palmioleophilaSW4’-W6 is used to decompose nitrite nitrogen (NO ₂-N) in the water. The strain of Candida palmioleophilaSW4’-W6 is deposited at Bioresource Collection and Research Center of Taiwan with a deposit number BCRC 920125. A method for treating water in culture pond using strain of Candida palmioleophilaSW4’-W6 is also disclosed.

Description

Glyphosate-resistant bacteria SW4'-W6 and its method for treating aquaculture water

The present invention relates to a kind of glyphosate-resistant bacteria, especially a kind of glyphosate-resistant bacteria SW4'-W6 that can decompose nitrite nitrogen; the present invention also relates to treating aquaculture water with the glyphosate-resistant bacteria SW4'-W6. method.

Aquaculture water contains nitrite nitrogen (NO ₂ -N) and other inorganic nitrogen. When natural water bodies receive wastewater containing inorganic nitrogen, it will cause the phenomenon of eutrophicatyion, which will make the water in the water body. The rapid proliferation of algae and plankton reduces the dissolved oxygen (DO) in the water body and deteriorates the water quality, resulting in a large number of deaths of fish and other aquatic organisms.

In view of this, it is indeed necessary to provide a glyphosate-resistant bacteria SW4'-W6 that can decompose nitrite nitrogen.

In order to solve the above-mentioned problems, the purpose of the present invention is to provide a kind of glyphosate-resistant bacteria SW4'-W6, which can be used for decomposing nitrite nitrogen in aquaculture water.

The second object of the present invention is to provide a method for treating aquaculture water, using the aforementioned glyphosate-resistant bacteria SW4'-W6.

The glyphosate-resistant bacteria SW4'-W6 that can decompose nitrite nitrogen of the present invention are deposited in the Food Industry Development Research Institute of the Republic of China, and the deposit number is BCRC 920125; the glyphosate-resistant bacteria SW4'-W6 have The ability to decompose nitrite nitrogen in wastewater is the effect of the present invention.

Based on the same technical concept, the method for treating aquaculture water of the present invention may include: providing aquaculture water containing nitrite nitrogen; and adding the aforementioned glyphosate-resistant bacteria SW4'-W6 into the aquaculture water , under the temperature of 20～35 ℃, make the glyphosate-resistant bacteria SW4'-W6 decompose the nitrite nitrogen in the culture water; in this way, by the biological activity of the glyphosate-resistant bacteria SW4'-W6, the Effectively decomposing the nitrite nitrogen in the breeding water and converting it into biomass that can be used by itself is the effect of the present invention.

The method for treating aquaculture water of the present invention, wherein, 10 ⁵ to 10 ⁷ CFU of glyphosate-resistant bacteria SW4'-W6 can be added to each liter of aquaculture water; The amount of bacteria is adjusted so that the glyphosate-resistant bacteria SW4'-W6 can effectively decompose the nitrite nitrogen in the aquaculture water.

The method for treating aquaculture water of the present invention may further comprise: detecting the nitrogen content in the aquaculture water, and then adding a carbon source to the aquaculture water so that the carbon-nitrogen ratio in the aquaculture water is between 15 and 30; for example , the carbon source can be glucose or sucrose; in this way, by adjusting the carbon-nitrogen ratio, the glyphosate-resistant bacteria SW4'-W6 can effectively decompose the nitrite nitrogen in the aquaculture water.

In the method for treating aquaculture water of the present invention, the salinity of the aquaculture water can be between 0 and 35‰; in this way, by adjusting the salinity, the glyphosate-resistant bacteria SW4'-W6 can be effectively Decompose the nitrite nitrogen in the culture water.

In order to make the above-mentioned and other objects, features and advantages of the present invention more obvious and easy to understand, the preferred embodiments of the present invention are exemplified below, and are described in detail as follows in conjunction with the accompanying drawings:

The glyphosate-resistant ( Candida palmioleophila ) SW4'-W6 system of the present invention is selected from the culture pond water of white shrimp with a salinity of 20‰, which is deposited in the food industry development of the Republic of China on December 01, 2020 Institute (the deposit number is BCRC 920125), and has the ITS (internally transcribed spacer) sequence shown in SEQ ID NO: 1, the glyphosate-resistant bacteria SW4'-W6 line has the ability to decompose nitrite nitrogen, so It can be used to decompose nitrite nitrogen in aquaculture water.

Generally speaking, workers can preferentially cultivate the glyphosate-resistant bacteria SW4'-W6 with a liquid fertilizer, and cultivate the glyphosate-resistant bacteria SW4'-W6 to a stationary phase, for example, at 20-35°C. At the same temperature, the glyphosate-resistant bacteria SW4'-W6 was cultivated to a concentration of 10 ⁷ CFU/mL, so that the glyphosate-resistant bacteria SW4'-W6 had good biological activity. In this embodiment, the formula of the liquid fertilizer can be as shown in Table 1 below.

Table 1. Formula of liquid fertilizer added amount molasses 17.25 grams Urea 1 gram distilled water Refill to 1 liter

Next, the worker can add the glyphosate-resistant bacteria SW4'-W6 into a culture water (preferably, it can be dispersed and sprinkled near the aerator), so that each liter of culture water can be mixed with 10 ⁵ -10 ⁷ CFU of anti-grass Glyphosate bacteria SW4'-W6, so the glyphosate-resistant bacteria SW4'-W6 can continue to grow nitrite nitrogen in the aquaculture water as a nutrient, and carry out biological metabolism to decompose the nitrite in the aquaculture water. nitrogen.

Before adding the glyphosate-resistant bacteria SW4'-W6 to the aquaculture water, preferably, the nitrogen content in the aquaculture water can be detected, and then a carbon source is added to the aquaculture water, so that the carbon-nitrogen ratio in the aquaculture water is between 15 ～30, so that the carbon source can also become the nutrient of the glyphosate-resistant bacteria SW4'-W6, so that the glyphosate-resistant bacteria SW4'-W6 can effectively decompose the nitrite nitrogen in the culture water; In this embodiment, the carbon source can be selected as cheap and easy-to-obtain glucose or sucrose, so that the cost of treating the aquaculture water can be reduced.

In addition, since the glyphosate-resistant bacteria SW4'-W6 can survive in aquaculture water with a salinity between 0 and 35‰, the glyphosate-resistant bacteria SW4'-W6 can be used to treat aquaculture freshwater Aquaculture water for aquatic or marine aquatic organisms.

In order to prove that the glyphosate-resistant bacteria SW4'-W6 are indeed new strains belonging to the glyphosate-resistant bacteria, and the glyphosate-resistant bacteria SW4'-W6 can effectively decompose nitrite nitrogen in water, the following experiments were carried out:

(A) Mycological characteristics of glyphosate-resistant bacteria SW4'-W6

The Gram-stained micrographs of the glyphosate-resistant bacteria SW4'-W6 are shown in Figure 1, and the cells have a spherical appearance.

The phylogenetic tree diagram as shown in Figure 2 can be drawn by the neighbor-joining method, showing that the glyphosate-resistant bacteria SW4'-W6 are new strains.

(B) Growth environment test of glyphosate-resistant bacteria SW4'-W6

The glyphosate-resistant bacteria SW4'-W6 were cultured in solid media at different temperatures, and the results were shown in Table 2. At a low temperature of 10°C or a high temperature of 50°C, the glyphosate-resistant bacteria SW4'- The growth of W6 was stagnant. At the temperature of 30℃, the glyphosate-resistant bacteria SW4'-W6 had the best growth condition.

Table 2. Growth rates at different temperatures 10℃ 20℃ 25℃ 28℃ 30℃ 35℃ 40℃ 50℃ Glyphosate-resistant bacteria SW4'-W6 - + ++ ++ +++ ++ ++ -

In addition, the glyphosate-resistant bacteria SW4'-W6 was cultured in solid medium with different salinities. The growth of W6 was stagnant, and it had a good growth condition under the salinity of 5～45‰.

Table 3. Growth rates at different salinities 5‰ 35‰ 45‰ 60‰ 70‰ 100‰ 135‰ 150‰ Glyphosate-resistant bacteria SW4'-W6 ++++ ++++ ++++ +++ +++ ++ + -

(C) Biosafety Assessment

In addition, the glyphosate-resistant bacteria SW4'-W6 were added to the culture water in which different aquatic organisms were cultured (the concentration of the glyphosate-resistant bacteria SW4'-W6 was 10 ⁶ CFU/mL), so that the aquatic organisms were cultured in the cultured water. After immersion in water for 7 days, the results showed that the glyphosate-resistant bacteria SW4'-W6 were resistant to yellowfin grouper ( Epinephelus flavocaeruleus ), fine point grouper ( Epinephelus cyanopodus ), milkfish ( Chanos chanos ), European common carp ( Cyprinus carpio ), Guppies ( Poecilia reticulata ), Catfish ( Hypostomus plecostomus ), tilapia ( Oreochromis sp. ), Red Broadband Butterfly ( Tropheus moorii ), Cyrtocara moorii , Cichlids ( Asistochromis christyi ) and other fish; white prawn ( Litopenaeus vannamei ), corrugated lobster ( Panulirus homarus ), grass shrimp ( Penaeus monodon ), red crayfish ( Cherax quadricarinatus ) and other shrimp; and ring clams ( Cyclina sinensis ), Portugal Mollusks such as the long oyster ( Magallana gigas ) are not biotoxic.

(D) Effects of different salinity in culture water

In this experiment, aquaculture water (group D1, D2, D3) with salinity of 0, 20 and 35‰ was selected for testing. Glucose was added to the culture water of each group to make the carbon-nitrogen ratio of the culture water 15. The glyphosate-resistant bacteria SW4'-W6 were made to contain 10 ⁷ CFU of glyphosate-resistant bacteria SW4'-W6 per liter of aquaculture water, and at 0, 24 and 48 hours, the levels of the culture water in each group were measured respectively. Nitrite nitrogen content.

Please refer to Figure 3. After 24 hours, the culture water of groups D1 and D2 with salinity of 0 or 20‰ did not contain nitrite nitrogen, while after 48 hours, the water of group D1 and D2 with salinity of 35‰ The culture water of group D3 still contained nitrite nitrogen with a concentration of about 0.26 mg/L.

(E) Effects of different carbon sources in aquaculture water

In this experiment, aquaculture water with salinity of 0‰ was selected for testing. Glucose or sucrose (groups E1 and E2) were added to the aquaculture water to make the carbon-nitrogen ratio of the aquaculture water 15. Then the glyphosate-resistant water was added. Bacteria SW4'-W6 were used to make each liter of culture water contain 10 ⁷ CFU of glyphosate-resistant bacteria SW4'-W6, and the content of nitrite nitrogen in the culture water of each group was measured at 0, 24 and 48 hours respectively. .

Please refer to Figure 4a, after 24 hours, the culture water of group E2 added with sucrose has no nitrite nitrogen, while the culture water of group E1 added with glucose is free of nitrite nitrogen after 48 hours .

In addition, in this experiment, aquaculture water with a salinity of 35‰ was used for testing. Glucose or sucrose (groups E3 and E4) were added to the aquaculture water to make the carbon-nitrogen ratio of the aquaculture water 15, and the anti-grass was added. Glyphosate SW4'-W6, make each liter of culture water contain 10 ⁷ CFU of glyphosate-resistant bacteria SW4'-W6, and measure the nitrous acid in each group of culture water at 0, 24, 48 and 72 hours respectively Nitrogen content.

Please refer to Figure 4b, after 48 hours, the culture water of group E2 added with sucrose has no nitrite nitrogen, and after 72 hours, the culture water of group E1 added with glucose has no nitrite nitrogen nitrogen.

(F) Effects of different carbon-nitrogen ratios in aquaculture water

In this experiment, aquaculture water with salinity of 0‰ was selected for testing, and the carbon-nitrogen ratio of the aquaculture water was adjusted to 5, 10, 15 or 30 (groups F1, F2, F3, F4) with sucrose, and the Glyphosate-resistant bacteria SW4'-W6, so that each liter of culture water contains 10 ⁷ CFU of glyphosate-resistant bacteria SW4'-W6, and measured at 0, 24, 48 and 72 hours in each group of culture water. Nitrite nitrogen content.

Please refer to Figure 5a, after 48 hours, the culture water of group F4 with a carbon-nitrogen ratio of 30 has no nitrite nitrogen, and after 72 hours, the culture water of group F3 with a carbon-nitrogen ratio of 15 has no nitrite nitrogen. Does not contain nitrite nitrogen.

In addition, in this experiment, aquaculture water with salinity of 35‰ was used for testing. After adjusting the carbon-nitrogen ratio of the aquaculture water to 5, 10, 15 or 30 with sucrose (groups F5, F6, F7, and F8), The glyphosate-resistant bacteria SW4'-W6 were added to make each liter of culture water contain 10 ⁷ CFU of glyphosate-resistant bacteria SW4'-W6, and the culture of each group was measured at 0, 24, 48 and 72 hours. The content of nitrite nitrogen in water.

Please refer to Figure 5b. After 48 hours, the culture water of groups F7 and F8 with carbon-nitrogen ratios of 15 and 30 did not contain nitrite nitrogen.

(G) Decomposition ability of ammonia nitrogen

The ability of the glyphosate-resistant bacteria SW4'-W6 to decompose ammonia nitrogen was also tested. In this experiment, aquaculture water (group D1, D2, D3) with salinity of 0, 20 and 35‰ was selected for testing. Glucose was added to the culture water of each group to make the carbon-nitrogen ratio of the culture water 15. The glyphosate-resistant bacteria SW4'-W6 were made to contain 10 ⁵ CFU of glyphosate-resistant bacteria SW4'-W6 per liter of culture water, and the ammonia nitrogen levels in the culture water of each group were measured at 0 and 24 hours respectively. content.

Please refer to Figure 6. After 24 hours, the culture water of Group G1, G2 and G3 with salinity of 0, 20 or 35‰ has no ammonia nitrogen.

To sum up, the glyphosate-resistant bacteria SW4'-W6 that can decompose nitrite nitrogen of the present invention have the ability to decompose nitrite nitrogen in wastewater, which is the effect of the present invention.

Furthermore, in the method for treating aquaculture water of the present invention, by virtue of the biological activity of the glyphosate-resistant bacteria SW4'-W6, the nitrite nitrogen in the aquaculture water can be effectively decomposed and converted into self-useable nitrites. The biomass (biomass) is the effect of the present invention.

Although the present invention has been disclosed by the above-mentioned preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes and modifications relative to the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the patent application attached hereto.

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[Fig. 1] In test (A), Gram-stained micrographs of glyphosate-resistant bacteria SW4'-W6. [Fig. 2] In test (A), the tree diagram of the relationship between the ITS sequences of the glyphosate-resistant bacteria SW4'-W6 and other strains of the genus Candida. [Figure 3] In test (D), the bar graph of the ability of glyphosate-resistant bacteria SW4'-W6 to decompose nitrite nitrogen in culture water with different salinities ( p < 0.05). [Figure 4a] In test (E), the bar graph of the ability of glyphosate-resistant bacteria SW4'-W6 to decompose nitrite nitrogen in culture water with salinity of 0‰ and adding different types of carbon sources ( p < 0.05). [Figure 4b] In test (E), the bar graph of the ability of glyphosate-resistant bacteria SW4'-W6 to decompose nitrite nitrogen in culture water with salinity of 35‰ and adding different types of carbon sources ( p < 0.05). [Picture 5a] In experiment (F), in culture water with salinity of 0‰ and different carbon-nitrogen ratios, the bar graph of the ability of glyphosate-resistant bacteria SW4'-W6 to decompose nitrite nitrogen ( p < 0.05). [Picture 5b] In experiment (F), in culture water with salinity of 35‰ and different carbon-nitrogen ratios, the bar graph of the ability of glyphosate-resistant bacteria SW4'-W6 to decompose nitrite nitrogen ( p < 0.05). [Figure 6] In test (G), the bar graph of the ability of glyphosate-resistant bacteria SW4'-W6 to decompose ammonia nitrogen in culture water with different salinities ( p < 0.05).

Domestic storage information Glyphosate-resistant bacteria SW4'-W6: TW Republic of China, Food Industry Development Research Institute of the Republic of China, 12/01/2020, BCRC 920125.

Claims (6)

A glyphosate-resistant bacteria SW4'-W6 that can decompose nitrite nitrogen is deposited in the Food Industry Development Research Institute of the Republic of China, and its deposit number is BCRC 920125. A method for treating aquaculture water, comprising: providing a culture water containing nitrite nitrogen; and The glyphosate-resistant bacteria SW4'-W6 according to claim 1 is added to the aquaculture water, and at a temperature of 20 to 35°C, the glyphosate-resistant bacteria SW4'-W6 is allowed to decompose the nitrite nitrogen in the aquaculture water. The method for treating aquaculture water according to claim 2, wherein 10 ⁵ to 10 ⁷ CFU of glyphosate-resistant bacteria SW4'-W6 is added to each liter of aquaculture water. The method for treating aquaculture water as claimed in claim 2, further comprising: detecting the nitrogen content in the aquaculture water, and then adding a carbon source to the aquaculture water so that the carbon-nitrogen ratio in the aquaculture water is between 15 and 30. The method for treating aquaculture water according to claim 4, wherein the carbon source is glucose or sucrose. The method for treating aquaculture water according to any one of claims 2 to 5, wherein the salinity of the aquaculture water is between 0 and 35‰.

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