CN113249272B

CN113249272B - Novel marine-source halotolerant zoogloea forming strain and application thereof

Info

Publication number: CN113249272B
Application number: CN202110724565.4A
Authority: CN
Inventors: 邱东茹; 李书杨; 刘双元
Original assignee: Institute of Hydrobiology of CAS
Current assignee: Institute of Hydrobiology of CAS
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2022-05-27
Anticipated expiration: 2041-06-28
Also published as: CN113249272A

Abstract

The invention provides a bryococcus of marine originTessaracoccus nanhaienisSYL-2 is a new strain of fungus, has obvious differences in 16S rRNA affinity, physiological and biochemical characteristics, BioLog GEN III determination and the like compared with the reported strain and other published bacterial strains, is a new strain of fungus, and has a preservation number of CCTCC NO: m20191064, the strain has better salt-tolerant characteristic and stable zoogloea forming ability, and has great potential in the aspect of high-salt sewage treatment.

Description

Novel marine-source halotolerant zoogloea forming strain and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to separation and identification of a new species of marine-derived halotolerant zoogloea forming bacteria and application of the new species in sewage treatment.

Background

In recent years, with the proliferation of population and the aggravation of natural ecological balance by activities, a plurality of malignant results of destroying the ecological environment are caused, and serious pollution of the water body environment, especially industrial waste water, is one of the types. Industrial wastewater mostly comes from industries such as chemical industry, agriculture, medicine and the like; in industrial production, a large amount of water resources are consumed, and in order to reduce the consumption of the water resources, a method of recycling the water resources is usually adopted in the industry, so that high salt water is formed; generally speaking, the industrial high-salt water has the characteristics of large discharge amount, wide source, high salt content, complex components and large difference of high-salt water generated in different industries. In the preparation process of medicines, pesticides and intermediates thereof, the processes of salting out, chemical synthesis, acid-base neutralization and the like can generate wastewater with high salt content, most of the wastewater has the characteristic of high salinity, and the salinity content of the wastewater is more than 10000 mg/L. Besides organic pollutants, the wastewater also contains a large amount of soluble inorganic salt ions such as calcium, magnesium, sodium, chlorine, sulfate radicals and the like and drug residues, and even contains radioactive substances, and in addition, after untreated high-salinity wastewater enters underground water, the hardness of underground water is increased, so that great force is generated on the daily life and the health of people. If the water with high salinity is drunk for a long time, teeth can be damaged, the health of human bodies can be harmed, and diseases such as kidney stones, gall-stones and the like can be caused seriously; therefore, the harm of the high-salinity wastewater to the life of people is not negligible. At present, the traditional domestic and foreign high-salinity wastewater treatment methods mainly comprise a physical method, a chemical method, a physical-chemical method, an artificial wetland treatment method and a biological method. The biological method is to treat pollutants contained in the industrial high-salinity wastewater through microbial flora, the treatment cost is relatively low, but the conditions are harsh, the salinity cannot be too high, otherwise the tolerance range of the microorganisms is exceeded, and particularly the salt tolerance of bacteria formed by zoogloea is generally poor, so that the treatment effect is greatly reduced. Therefore, the effect of the salt-tolerant zoogloea forming bacteria on improving the effect of the activated sludge method on high-salt sewage treatment is huge.

The marine fishes are deeply loved by people due to high nutritional value and delicious taste, along with the rapid development of economy and the increasing of human demand, people have higher requirements on the types, quality and demand of marine products, the marine products in coastal regions are frequently fished, the marine fishery resources are exhausted due to the reasons of transitional fishing, seawater pollution, resource consumption and the like, but the marine fishes are higher in price in the market due to the factors of reduced fishing amount, low supply amount, difficult cultivation, high cultivation cost and the like, so that the improvement of the living standard of people is not facilitated, and the sustainable development of the marine fishery is limited. The mariculture is suitable for the life, the mariculture industry is developed vigorously due to the advantages of high culture efficiency, small occupied area, diversified modes, water resource saving and the like, the demand is increased, the mariculture industry in China is also changed into an intensive, high-density and high-yield mode, but the abuse of bait throwing, discharged excrement and fish drugs directly or indirectly influences the water quality balance in the high-density and intensive culture mode. Ammonia nitrogen, nitrite, sulfide, COD and the like are common harmful substances in the culture seawater. At present, chemical, physical and biological methods are mainly used for removal.

The invention separates and identifies a new species (Tessaracococcus nanhaiienis) SYL-2 of the bryococcus of marine origin, and the strain has better salt-resistant property and stable zoogloea forming capability, so the strain has great potential in the aspects of high-salt chemical wastewater, medical wastewater treatment, marine product culture and the like.

Disclosure of Invention

The invention provides a new species Tessaracococcus nanhaiienis SYL-2 of marine origin bryococcus, the strain has better salt resistance and stable zoogloea forming capability, and has great potential in the aspect of sewage treatment (especially high-salt sewage).

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention separates a strain SYL-2 which can stably form a zoogloea from an activated sludge sample, compared with the reported strains, the strain has obvious differences in the aspects of 16S rRNA affinity, physiological and biochemical characteristics and the like, is a new strain, is named as a bryococcus Tessarcacus nanhaiienis SYL-2, is preserved in China center for type culture collection in 2019 in 12 months and 18 months, and has the preservation number of CCTCC NO: m20191064, accession number: wuhan university in Wuhan, China.

The salt tolerance of the rhodococcus narhaiienis SYL-2 is 0-6%, and compared with other zoogloea forming bacteria, the rhodococcus narhaiienis obviously higher in salt tolerance and better in treatment effect on sewage (particularly high-salt sewage).

Compared with the prior art, the invention has the following beneficial effects:

the marine-derived bryococcus narhaenis SYL-2 is a new strain, has obvious differences in 16S rRNA affinity, physiological and biochemical characteristics compared with the reported strain and other published bacterial strains, and is a new strain. The strain provides a good research material for researching the salt tolerance relationship between the zoogloea and bacteria, the bacterium formation salt tolerance mechanism of the zoogloea and the like, and the strain also has application potential in the aspects of high-salt chemical industry sewage, medical wastewater treatment, marine product culture and the like.

Drawings

FIG. 1 shows a biological evolutionary tree constructed by a Bryococcus tessaracacus nanhaiienis SYL-2 based on 16S rRNA gene.

FIG. 2 is a Biolog Gen III carbon source utilization picture and a comparison chart of Tessacacoccus nanhaiienis SYL-2.

FIG. 3 is an optical microscope photograph of a Bryococcus narhaiienis SYL-2 grown in TSB medium for 32 hours.

FIG. 4 is a TEM photograph of a 32-hour growth of Tensaracoccus nanhaiienis SYL-2 in TSB medium.

FIG. 5 shows the morphology of the Bryococcus narhaiienis SYL-2 after 48h growth in TSB medium, standing and shaking.

FIG. 6 shows the purification of textile wastewater by the Bryococcus tessaracaccus nanhaiienis SYL-2 (before left treatment and after right treatment).

Detailed Description

The technical scheme of the invention is the conventional technology in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.

The culture medium used in the examples of the present invention was as follows:

formulation of R2A liquid medium (1L): 0.5g of tryptone, 0.5g of yeast extract, 0.5g of starch, 0.5g of acid hydrolyzed casein, 0.3g of sodium pyruvate, 0.3g of dipotassium phosphate and 0.024g of magnesium sulfate, and the pH value is adjusted to 7.0 +/-7.2;

formulation of R2A solid medium (1L): 0.5g of tryptone, 0.5g of yeast extract, 0.5g of starch, 0.5g of acid hydrolyzed casein, 0.3g of sodium pyruvate, 0.3g of dipotassium phosphate, 0.024g of magnesium sulfate and 15g of agar, and adjusting the pH value to 7.0 +/-7.2;

formulation (1L) of liquid seed culture set (TSB): tryptone 17g, soybean peptone 3g, D-glucose 2.5g, NaCl 5g, K₂HPO₄ 2.5g，pH 7.3±0.2；

Formulation of solid seed medium (TSBA) (1L): tryptose peptone 17g, soytone 3g, D-glucose 2.5g, NaCl 5g, K₂HPO₄2.5g, pH 7.3 +/-0.2 and 15g of agar.

The present invention is described in detail below with reference to examples, which will assist those skilled in the art in further understanding the present invention, but are not intended to limit the present invention in any way.

Example 1 isolation and characterization of Rhodococcus narhaiienis SYL-2:

activated sludge samples were collected from a sewage treatment plant in hong Kong and graded with sterile water in a clean bench (10)^-l、10^-2、10^-3、10^-4、10^-5) Diluting, sucking 100 μ l of the diluent, spreading on a solid plate of R2A culture medium, and culturing at 28 deg.C for 48 h; selecting a solid plate, and inoculating the single clone on the solid plate into a centrifugal tube of 1.5ml to culture by using an R2A liquid culture medium; screening strains capable of stably forming zoogloea, transferring and expanding the strains into a 10ml centrifugal tube, culturing the strains in an R2A liquid culture medium, and screening and expanding strains capable of still stably forming zoogloea; and (3) sucking the 10 mu l of the bacterial liquid to an R2A solid plate, carrying out streak purification, culturing at 28 ℃ for 48h, carefully observing the colony unicity of the plate, selecting a single clone, inoculating the single clone to a 12ml glass culture tube, and carrying out amplification culture to obtain a purified strain SYL-2.

The cells were collected by centrifugation, the genome was extracted, the 16SrRNA gene sequence of the strain SYL-2 was amplified and sequenced using published 16S universal primers (27F and 1492R, primer sequences 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'), and the PCR amplification product was preliminarily sequenced by Scobis Okagaku corporation (TsingKe Bio). The sequencing sequence is subjected to blastn comparison at NCBI, and finally the highest homology of the strain SYL-2 and a near source model strain is determined to be 95.66%, and the 16S rRNA of the strain has lower than 97% homology with a known database and is more likely to be a new bacterial species.

To further confirm, the construction of the pMD-18T vector was performed to confirm sequencing and new species identification. Specifically, the upstream and downstream homologous arm sequences (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3') are respectively amplified by using high fidelity enzyme, the amplified sequences are connected with a pMD-18T vector, and the recombinant vector enters DH5 alpha chemical competent cells through heat shock transformation; DH 5. alpha. containing the recombinant vector was recovered at 37 ℃ and then plated on LB solid plate containing ampicillin; selecting colonies from an LB solid plate as a template to carry out colony PCR amplification, selecting clones with correct amplification fragment size to carry out liquid culture, and sequencing the quality-improved particles (M13-47: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'; M13-48: 5'-GAGCGGATAACAATTTCACACAGG-3'); the plasmid sequencing tailoring results were subjected to blastn alignment at NCBI and found to have the highest sequence homology of the 16S rRNA gene with tessaraccus arenae strain CAU 1319(NR156959) and a similarity of 95.85% (interspecies distribution less than 97%). The 16S rRNA gene is used for constructing a phylogenetic tree (figure 1), and compared with other clades, the strain SYL-2 and the Tessaracoccus clade are one branch, wherein the strain is most similar to the Tessaracoccus arenae CAU 1319 model strain. Sequencing the whole genome of the strain SYL-2, and performing average nucleotide homology (ANI) analysis and DNA-DNA molecular hybridization (DDH) on the whole genome of the strain SYL-2 and the most similar 3 model strains by using a server EzBioCloud to obtain the strain SYL-2 with the ANI value of 75.23-77.46% (interspecies distribution 65-90%) and the DDH value of 19.2-26.1% (interspecies distribution less than 50%); therefore, the strain SYL-2 is a novel bacterium of the genus Tessaracococcus.

Compared with the reported strains and other published bacterial strains, the strain SYL-2 has obvious differences in 16S rRNA affinity, physiological and biochemical characteristics, and is a new strain. Biolog Gen iii carbon source utilization physiological and biochemical identification of SYL-2 as shown in fig. 2; SYL-2 grows on nutrient agar (TSBA) culture medium to form circular colony, and after 2 days of culture on TSB agar solid culture medium, the diameter of the monoclonal colony is about 0.8-2.1mm, and the colony is semitransparent milky white, circular, smooth, flat and slightly convex and has complete edges. The bacteria is gram negative, single, paired or piled, can form stable zoogloea and spherical new bacteria without spore formation, and the diameter of the bacteria is about 0.9-2.1 mu m. Non-hydrolyzed gelatin, arginine, alanine, glutamic acid, histidine, serine, etc.; dextrin, maltose, trehalose, cellobiose, sucrose, stachyose, glucose, pectin and the like can be used as a unique carbon source for growth; the optical micrograph is shown in FIG. 3, and the transmission electron micrograph is shown in FIG. 4; the morphology in the liquid culture glass tube is shown in FIG. 5.

In conclusion, the strain SYL-2 belongs to a new species of Tessaracococcus, and is named as a new marine-source bacterium Tessaracococcus nanhaiienis SYL-2 by the molecular microorganism of the institute of aquatic organisms of Chinese academy of sciences, a Tuotus researchers in a biotechnology laboratory and Robos littora rooting according to the international double name method (international naming convention: genus name + species name), genus name Tessaracococcus nanhaiienis, species name nanhaiienis and number SYL-2. Tessaracococcus nanhaiienis SYL-2 has been preserved in the China center for type culture Collection in 2019, 12 and 18 months with the preservation number of CCTCC NO: m20191064, accession number: wuhan university in Wuhan, China.

Example 2 comparison of salt tolerance of different zoogloea-forming bacteria

In order to compare the salt tolerance of different zoogloea forming bacteria, the salt tolerance interval is determined by changing the salinity of the R2A liquid culture medium. Selecting a monoclonal colony on an R2A plate, inoculating the colony into a conical flask filled with 50mL of R2A liquid culture medium for culture, shaking and culturing for about 2 days at the optimal growth temperature, observing that when the colony is approximately in a logarithmic growth phase, sucking fresh bacterial liquid according to 1 percent (the influence on the salinity is not enough to influence the experimental result) of inoculation quantity, transferring the fresh bacterial liquid into 100mL of prepared R2A liquid culture medium with the salinity of 0-15 percent NaCl (w/v, the concentration gradient is 0.5 percent), shaking and culturing for 3-4 days according to the determined optimal growth temperature, then centrifugally collecting thalli, weighing the wet weight by an electronic balance, and determining the salt-tolerant interval according to the wet weight of the thalli. The salt tolerance of different zoogloea forming bacteria is shown in table 1.

TABLE 1 comparison of salt tolerance of different zoogloea-forming bacteria

The salt tolerance of the strain SYL-2 is 0-6%, and compared with other zoogloea forming bacteria, the salt tolerance is obviously stronger, and the strain SYL-2 has development potential in high-salt sewage treatment with the salinity lower than 6%.

Example 3 removal of COD from high salinity high wastewater by Tessaracococcus nanhaienis SYL-2

We collected textile wastewater from Wuhan Yanghuan textile mill and measured that its salinity was about 3.65% and its COD was about 373.76 mg/L. Selecting a bacterial strain Tessaracococcus nanhaiienis SYL-2 monoclonal, culturing in 2ml of R2A liquid culture medium for 48h, washing twice with sterilized double distilled water, adding into 200ml of textile sewage, and performing shake culture for 24 h. The strain Tessaracococcus nanhaiienis SYL-2 is found to have an obvious purification effect on textile sewage, the COD removal rate of the textile sewage exceeds 65%, and the textile sewage is obviously clarified after purification (see table 2 and figure 6). Table 2 shows the COD removal of textile wastewater by Tessaracococcus nanhaiienis SYL-2

Claims

1. Bryococcus (Moss) (M)Tessaracoccus nanhaienis) SYL-2, characterized in that the preservation number of the strain is CCTCC NO: m20191064.

2. The bryococcus of claim 1, (b) cTessaracoccus nanhaienis) Application of SYL-2 in sewage treatment.