Rhodococcus coryneform strain and application thereof

Abstract

The invention relates to the technical field of micro-ecology, in particular to a rhodococcus coryneform strain and application thereof in aquaculture. The invention discloses a strain of Rhodococcus corynobacteria, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.23799. The invention further discloses a preparation method of the strain of Rhodococcus corynobacteria. The rhodococcus strain can oxidize sulfides under the condition of low dissolved oxygen, does not reduce the pH value of a water body, reduces sulfide pollution of an aquaculture water body, relieves bottom mud acidification, and has no toxic effect on cultured animals.

Description

Technical Field

[0001] This invention relates to the field of microecological technology, specifically to a corynebacterium-like Rhodococcus strain and its applications. Background Technology

[0002] With the rapid development of aquaculture, the problem of environmental degradation in aquaculture has intensified, leading to frequent outbreaks of diseases in economically important species. Excessive nutrient input is a common strategy in aquaculture to increase production. Uneaten high-protein feed and excrement settle at the bottom of the ponds, resulting in a generally high nutrient load and low dissolved oxygen levels in the aquaculture system. Under low dissolved oxygen conditions, microorganisms incompletely decompose organic matter, producing organic acids that acidify the pond bottom sediment, affecting the release and utilization of nutrients.

[0003] Investigations have revealed severe sulfide pollution in the water and sediment environments of many aquaculture areas in my country, with hydrogen sulfide being particularly harmful to farmed animals and hindering the stable development of the aquaculture industry. Hydrogen sulfide is produced through two pathways: first, the decomposition of organic sulfides, such as the hydrolysis of proteins in uneaten feed into amino acids, which are then released as hydrogen sulfide by the enzymes of heterotrophic bacteria; second, the reduction of sulfates in the sediment into hydrogen sulfide by anaerobic bacteria.

[0004] When hydrogen sulfide is present in aquaculture ponds, fish exhibit symptoms similar to surfacing during the day. Turning on aerators does not alleviate this surfacing phenomenon; instead, the fish disperse, and the surfacing becomes more severe with increased aeration. Furthermore, a pungent, rotten egg-like odor can be detected downwind of the pond. Hydrogen sulfide, once absorbed into the fish's body, converts the low-valent iron ions in hemoglobin to high-valent iron ions, causing hemoglobin to lose its oxygen-carrying capacity, resulting in tissue hypoxia and, in severe cases, death. Therefore, preventing and reducing hydrogen sulfide levels is crucial for successful aquaculture.

[0005] Sulfur-oxidizing bacteria are a broad category of microorganisms capable of oxidizing and reducing sulfides. They can convert reduced sulfides into sulfates, inhibit the metabolism of sulfate-reducing bacteria, reduce sulfide pollution, and improve the aquaculture environment. Based on their oxidation pathways, they can be divided into acid-producing sulfur-oxidizing bacteria and alkali-producing sulfur-oxidizing bacteria. These microorganisms have significant value in industry and environmental protection. Current research mainly focuses on biometallurgy, treatment of heavy metals in sludge and wastewater, and coal desulfurization. Research on their application in aquaculture to reduce sulfide pollution is relatively limited. Attached Figure Description

[0006] Figure 1 Colony morphology of HQ40 after 24 hours of incubation on LB plates;

[0007] Figure 2 Gram staining morphology of HQ40 bacterial cells;

[0008] Figure 3The sulfur oxidation capacity of HQ40 and its effect on the pH of the fermentation broth;

[0009] Figure 4 The effect of dissolved oxygen on the growth of HQ40;

[0010] Figure 5 The effect of dissolved oxygen on the sulfur oxidation capacity of HQ40;

[0011] Figure 6 The effect of HQ40 fermentation broth on the survival rate of Litopenaeus vannamei;

[0012] Figure 7 The effect of HQ40 on the survival rate of zebrafish;

[0013] Figure 8 Comparison of HQ40 fermentation broth's effect on the removal of sulfides from bottom sediment. Detailed Implementation

[0014] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0015] Example 1: Screening, isolation and identification of sulfur-oxidizing bacteria

[0016] Enrichment medium: 5g Na2S2O3, 1L aged seawater, sterilized at 121℃ for 15min, ready for use.

[0017] Screening medium: 5g Na2S2O3•5H2O, 0.2g MgCl2•6H2O, 1.2g K2HPO4, 1.2g KH2PO4, 0.4g NH4Cl•6H2O, 0.01g FeSO4•7H2O, 40g NaCl, 1L pure water, pH 6.8-7.0, sterilized at 121℃ for 15min, and 18g agar per liter of solids to prepare screening plates.

[0018] Differential medium: Phenol red (final concentration 0.004 g / L) was added to the screening medium.

[0019] LB medium: 10g peptone, 5g yeast extract, 10g NaCl, 1L pure water, sterilized at 121℃ for 15min, and 18g agar per liter of solids to make LB plates.

[0020] Iodometric method: Take 100 μL of the screening medium fermentation broth after inoculating the strain, dilute with water to 5 mL, add 200 μL of 17.5 mol / L acetic acid and 100 μL of 1% starch solution, mix well, and titrate with 0.01 mol / L iodine solution until a persistent blue color appears. Calculate S2O3 by the amount of iodine consumed in the titration. 2- The content of S2O3. Calculation formula: Y=(V-V0)×C×M×X / 50, Y: S2O3 content.2- The amount is expressed in g / L; C: the concentration of the iodine solution being titrated, which is 0.01 mol / L; M: S2O3 2- The molecular weight is 112 g / mol; X: dilution factor; V: volume of iodine solution consumed in titrating the sample; V0: volume of iodine solution consumed in titrating distilled water.

[0021] The formula for calculating oxidation rate is as follows: Oxidation rate = (Y 初 -Y 末 ) / Y 初 Y 初 The initial culture medium contained S2O3 2- quantity; Y 末 S2O3 calculated by iodometric method after fermentation culture 2- The amount.

[0022] Initial screening of strains: Water samples were collected from shrimp farming ponds in Fujian Province. Approximately 1 g of the sample was added to the enrichment medium and incubated at 28°C and 150 rpm for 3 days. Then, the enrichment medium was re-inoculated at a 10% inoculum, and this process was repeated 3 times. The final enrichment culture was diluted 10-fold, and different dilutions were spread onto screening plates. The plates were inverted and incubated at 28°C for 7 days. After 7 days, the purified strains were spread onto the plates.

[0023] Qualitative screening of strains: Single strains of purified bacteria were inoculated into differential culture medium and incubated upside down at 28°C for 7 days. The results were then observed. The blank medium without inoculation was light red. Acid-producing sulfur oxidizing bacteria would lower the pH of the medium and turn it yellow, while alkali-producing sulfur oxidizing bacteria would raise the pH and turn it dark red. Alkali-producing sulfur oxidizing bacteria were then screened and preserved.

[0024] Quantitative secondary screening of strains: After purifying the obtained alkali-producing sulfur-oxidizing bacteria by streaking on LB agar plates for three generations, the culture was inoculated into LB liquid medium as a seed culture and cultured at 28℃ and 150 rpm for 24 h. Then, the culture was inoculated into screening medium and cultured under the same conditions for 3 days. The concentration of thiosulfate in the fermentation broth was determined by iodometric titration, and the oxidation rate was calculated. The strain with the highest oxidation rate was identified.

[0025] Strain identification: The obtained strain was subjected to colony PCR, and the PCR product was sequenced to obtain the 16S rDNA sequence. The 16S rRNA sequence is shown in Table SEQ ID No. 1. The sequence was compared with gene sequences already registered in GenBank, and the results showed that it had 99.3% similarity to *Rhodococcoides corynebacterioides* strain SCG11, thus identifying it as *Rhodococcoides corynebacterioides* strain HQ40. Its morphology on LB agar plates is shown below. Figure 1As shown, the colonies are orange-red, approximately 1 mm in diameter, with neat edges, a raised surface, and are easy to pick up. Gram staining morphology is as follows: Figure 2 As shown, it is Gram-negative.

[0026] The obtained corynebacterioides HQ40 was deposited on November 15, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and classified as: Rhodococcoides corynebacterioides, with accession number CGMCC No. 23799.

[0027] Example 2: Sulfur oxidation capacity of HQ40

[0028] Single bacteria were inoculated into LB liquid medium and cultured at 150 rpm and 28°C for 24 h. Then, 3% inoculum was inoculated into selection medium and cultured at 150 rpm and 28°C for 4 consecutive days. The concentration of thiosulfate in the fermentation broth was measured daily by iodometric titration and the pH value of the fermentation broth was measured using a pH meter.

[0029] LB medium: 10g peptone, 5g yeast extract, 10g NaCl, 1L pure water, sterilized at 121℃ for 15min, and 18g agar per liter of solids to make LB plates.

[0030] Screening medium: 5g Na2S2O3•5H2O, 0.2g MgCl2•6H2O, 1.2g K2HPO4, 1.2g KH2PO4, 0.4g NH4Cl•6H2O, 0.01g FeSO4•7H2O, 40g NaCl, 1L pure water, pH 6.8-7.0, sterilized at 121℃ for 15min, and 18g agar per liter of solids to prepare screening plates.

[0031] The results are as follows Figure 3 The results showed that the sulfide concentration decreased from 24 hours, reaching its lowest value at 72 hours. At 72 hours, the sulfide oxidation rate was 90.21%. The pH of the fermentation broth increased as the sulfide concentration decreased, reaching 7.9 at 72 hours. The initial pH of the uninoculated blank medium was 6.9; therefore, compared to the uninoculated blank medium, the pH of the experimental group increased by 1 unit at 72 hours.

[0032] Example 3: Effect of dissolved oxygen on the sulfur oxidation capacity of HQ40

[0033] Single bacteria were inoculated into LB liquid medium and cultured at 150 rpm and 28℃ for 24 h. Then, 3% inoculum was inoculated into selection medium and cultured in shakers at speeds of 0, 40, 80, 120, 150 and 180 for 3 consecutive days. Equal amounts of fermentation broth were taken every 8 h to count the bacteria and determine the growth capacity of the strains under different dissolved oxygen conditions. At the same time, the concentration of sodium thiosulfate in the fermentation broth was determined by iodometric titration.

[0034] The results are as follows Figure 4 As shown, there was no significant difference in viable cell count and growth rate between 80-180 rpm. At 40 rpm, the lag phase was approximately 8 hours longer than at 80-180 rpm, and the viable cell count of the statically cultured strain was only 1% of that at 80-180 rpm. Sulfur oxidation was observed as follows... Figure 5 As shown, the sulfide concentration decreased to a similar degree when the rotation speed was 80-180 rpm, but at 40 rpm, the sulfide concentration began to decrease significantly at 48 hours. This indicates that HQ40 can tolerate a certain degree of low oxygen and can function effectively even at the bottom of ponds with low oxygen content.

[0035] Example 4: Effects of HQ40 on cultured organisms

[0036] Inoculate a single bacterium into LB liquid medium, incubate at 150 rpm and 28°C for 24 hours, then add to the aquaculture water to achieve a final bacterial concentration of 10. 5 CFU / ml, with the group without bacterial solution serving as a control, 50 juvenile Pacific white shrimp (initial body length 4.0±0.1cm) were stocked, with 3 replicates per group. The rearing tanks were 1m×1m in area and 0.7m deep. The shrimp were fed formulated feed at 5% of their body weight daily, and the water salinity was controlled at 1.8%-2.0%, pH 8.2-8.5, and water temperature 25-28℃. The survival rate of the reared animals was recorded after one week. Figure 6 As shown, the survival rate of shrimp larvae was 80%~86%, which was not significantly different from the survival rate of the control group, indicating that HQ40 has no toxic effect on farmed animals.

[0037] Example 5: Effects of HQ40 on cultured organisms

[0038] Inoculate a single bacterium into LB liquid medium, incubate at 150 rpm and 28°C for 24 hours, then add to the aquaculture water to achieve a final bacterial concentration of 10. 5 CFU / ml, with the untreated group serving as a control, 50 zebrafish (15 dpf) were stocked, with 3 replicates per group. The experimental pond had an area of ​​10cm × 10cm and a water depth of 6cm. Small amounts of brine shrimp were fed daily at regular intervals. The fish were cultured in a 28℃ incubator. The survival rate of the cultured animals was recorded after one week. Figure 7 As shown, the survival rate of zebrafish was 86%~90%, which was not significantly different from the survival rate of the control group, indicating that HQ40 has no toxic effect on farmed animals.

[0039] Experiment 1: Removal of sulfides from pond sediment by HQ40 fermentation broth

[0040] The interaction of sulfides with iron oxides and organic matter in the sediment leads to sediment blackening. Take 50g of blackened pond sediment and add it to an experimental bottle, cover with a 2cm water layer, and then add HQ40 fermentation broth to bring the final bacterial concentration to 10. 5 The concentration of cfu / ml was used as a control, with the group without bacterial solution serving as the reference. After standing at room temperature for 7 days, the color change of the sediment was observed. The results are as follows: Figure 7 The results showed that, compared with the group without bacterial solution, the sediment turned gray after adding bacterial solution, indicating that HQ40 fermentation broth can oxidize sulfides in the sediment and reduce sulfide deposition.

[0041] sequence list

[0042] SEQ ID No. 1:

[0043]

Claims

1. A strain of *Rhodococcus corynebacterioides*, characterized in that, Its accession number is CGMCC No. 23799.

2. The *Rhodococcus* strain resembling a corkyloidea according to claim 1, characterized in that, The 16S rRNA sequence of the *Rhodococcus pyogenes* strain is shown in Table SEQ ID No. 1, and the *Rhodococcus pyogenes* strain has alkalogenic sulfur oxidation function.

3. A microecological preparation containing the *Rhodococcus solani* strain as described in claim 1 or 2.

4. A method for degrading sulfides in aquaculture water or bottom sediment, characterized in that, Add the *Rhodococcus* strain of claim 1 or 2 or the microecological preparation of claim 3 to the water body or sediment.

5. The method according to claim 4, characterized in that, The viable count of *Rhodococcus quinquefolius* strains in the water or sediment was 10. 4 ~10 6 CFU / mL.

6. The method according to claim 5, characterized in that, The viable count of *Rhodococcus quinquefolius* strains in the water or sediment was 10. 5 CFU / mL.

7. The application of the *Rhodococcus* strain described in claim 1 or 2 in improving aquaculture water or acidified sediment environments.

8. The application of the *Rhodococcus* strain of claim 1 or 2 in the removal of sulfides from aquaculture water or sediment.

9. The use of the *Rhodococcus* strain of claim 1 or 2 in the preparation of formulations for improving aquatic environments.

10. A fermentation method for a *Rhodococcus* strain resembling a corkyloidae according to claim 1 or 2, characterized in that, Inoculate a single bacterium into LB liquid medium and incubate at 40-180 rpm for 24 hours at 28°C.

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