Methods for treating saline wastewater

Abstract

The present invention relates to a semi-continuous culture method capable of treating saline wastewater, wherein inorganic nutrients such as nitrogen salts and / or phosphorus salts are recovered by culturing halophilic microorganisms in saline wastewater. The method can not only treat saline wastewater on a large scale to make it meet the discharge standards, but the treated saline wastewater can also be used to produce table salt, making the semi-continuous culture method of the present invention highly cost-effective and economically beneficial.

Description

Technical Field

[0001] The present invention relates to the field of wastewater treatment, and in particular to a method for treating saline wastewater, which realizes large-scale recovery of inorganic nutrients such as nitrogen salts and / or phosphorus salts in saline wastewater by semi-continuously culturing halophilic microorganisms in the saline wastewater. Background Art

[0002] In the production process of various industries such as chemical industry, agriculture, textile industry and leather industry, saline wastewater is generated (Lefebvre and Moletta, 2006), and saline wastewater contains a variety of organic pollutants. Taking the processing water in the kelp production process as an example, the preservation of kelp requires the use of a large amount of salt (mainly sodium chloride) to extend the shelf life of kelp. Therefore, a large amount of high-salinity processing water is generated during the rinsing of kelp. At the same time, because the kelp undergoes high-temperature treatment during the rinsing process, the high-salinity processing water also contains a large amount of organic matter (mainly sodium alginate) and inorganic nutrients. If the high-salinity processing water containing a large amount of organic pollutants and inorganic nutrients is not effectively treated, it will cause great harm to the ecological environment of the water body, such as eutrophication of the water body. Therefore, before discharging this high-salinity saline wastewater, it is necessary to effectively remove or recover the organic pollutants and salts in it. According to Woolard and Irvine (1995), salinity in saline wastewater may exceed 15% (e.g., kelp processing water is almost saturated with sodium chloride), and the standard for defining high-salinity wastewater or brine is wastewater with a salinity exceeding 3.5% and containing organic matter.

[0003] The treatment of saline wastewater usually requires the combined application of multiple steps to ultimately achieve satisfactory wastewater treatment results. The main disadvantage of physical and chemical methods is the high operating costs caused by high-frequency equipment maintenance. Therefore, microbial methods, as a supplement or alternative to physical and chemical methods, have received increasing attention from scientific research and the business community. Therefore, there is still a need in the art for a method that can be scaled and efficiently used to treat saline wastewater. Summary of the invention

[0004] The object of the present invention is to provide a semi-continuous culture method capable of treating saline wastewater, which realizes the recovery of inorganic nutrients such as nitrogen salts and / or phosphorus salts in saline wastewater by culturing halophilic microorganisms in saline wastewater. The method can not only treat saline wastewater on a large scale to make it meet the discharge standards, but also produce table salt from the saline wastewater, thereby realizing the scale benefit and economic benefit of treating saline wastewater.

[0005] The inventors have achieved large-scale treatment of high-salinity saline wastewater by using a Dunaliella sp. strain obtained by culturing and separating and purifying the kelp production and processing water sampled from Shandong Haizhibao Marine Technology Co., Ltd. (hereinafter referred to as "Haizhibao Company") and a microbial combination including the strain and its symbiotic bacteria in a semi-continuous culture method. Thus, the present invention has been completed.

[0006] Therefore, in a first aspect, a method for treating saline wastewater is provided, comprising: semi-continuously culturing halophilic microorganisms in the saline wastewater; wherein the halophilic microorganisms are Dunaliella sp. or a microbial community including the Dunaliella sp.; and wherein the Dunaliella sp. is an algae strain preserved on October 14, 2020 at the Guangdong Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with a preservation number of GDMCC No: 61230.

[0007] In a second aspect, the present invention provides a method for producing edible salt, the method comprising:

[0008] (a) implementing the method described in the first aspect to obtain treated saline wastewater;

[0009] (b) evaporating the treated saline wastewater to obtain edible salt.

[0010] In summary, the present invention provides a large-scale method capable of treating saline wastewater by using Dunaliella salina that can adapt to a high-salinity environment or a microbial community comprising the Dunaliella salina and its symbiotic bacteria in a semi-continuous culture method. The inventors of the present invention have achieved the maximum recovery efficiency of inorganic nutrients and the accumulation of photosynthetic pigments in saline wastewater by optimizing the semi-continuous culture method, providing a more environmentally friendly, cost-effective, and sustainable new approach for the treatment of saline wastewater, and providing feasibility for subsequent large-scale applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The technical solutions and benefits of the present invention will become apparent to those skilled in the art with the benefit of the following detailed description and with reference to the accompanying drawings.

[0012] Figure 1 The separation and purification of Dunaliella salina and symbiotic bacteria from the high-salinity process water of Haizhibao Company according to one embodiment of the present invention is shown.

[0013] Figure 2 Microscope images (60x objective) of (A) and (B) Dunaliella salina and (C) and (D) symbiotic bacteria separated from high-salinity process water of Haizhibao Company according to one embodiment of the present invention are shown.

[0014] Figure 3 Shown are (A) an electrophoretic diagram of ITS PCR amplification products of Dunaliella salina according to one embodiment of the present invention; and (B) an electrophoretic diagram of 16S rDNA PCR amplification products of symbiotic bacteria.

[0015] Figure 4 The figures show the batch culture of Dunaliella salina in universal culture medium of different salinities according to one embodiment of the present invention, wherein: (A) first batch culture; (B) second batch culture; (C) specific growth rate. Three biological replicates were set for each treatment.

[0016] Figure 5 The present invention shows a batch culture of Dunaliella salina in synthetic process water according to one embodiment of the present invention, wherein: (A) growth curve; (B) nitrogen salt (NH4 + ) and (C) phosphate (PO4 3+ ) residual concentration. NH4 + and PO4 3+ The detection limits of 10 μg / L and 10 μg / L were 0.4 mg / L and 0.1 mg / L, respectively. Three biological replicates were set for each treatment.

[0017] Figure 6 The present invention shows the treatment of synthetic process water by batch culture of Dunaliella salina in a bag bioreactor according to one embodiment of the present invention, wherein: (A) experimental device picture; (B) growth curve; (C) biomass accumulation. Alginate+ / - respectively represents the presence / absence of alginate in the process water. All measurements were repeated three times.

[0018] Figure 7 The figure shows the change of photosynthetic pigment concentration of Dunaliella salina during batch culture according to one embodiment of the present invention. Alginate+ / - represents the presence / absence of sodium alginate in the process water, respectively. Chl a / b and carotenoids represent chlorophyll a / b and total carotenoids, respectively. All measurements were repeated three times.

[0019] Figure 8 The semi-continuous culture of Dunaliella salina in a bag reactor according to one embodiment of the present invention is shown, wherein: (A) the color change of the algae solution during the semi-continuous culture; (B) the growth curve, the OD is measured before and after the process water is replaced 725 ; (C) Biomass accumulation, biomass concentration (dry weight) was measured before changing the process water. All measurements were repeated three times.

[0020] Fig. 9Shown is the residual nutrient salt concentration in the synthetic process water during semi-continuous culture according to one embodiment of the present invention. Samples were taken to measure the concentration of ammonia nitrogen and active phosphoric acid (in terms of phosphorus element) before changing the process water every day. The measurement limits of ammonia nitrogen and active phosphoric acid (in terms of phosphorus element) were 0.4 mg / L and 0.1 mg / L respectively. All measurements were repeated three times.

[0021] Fig.10 The concentration changes of photosynthetic pigments during semi-continuous culture according to one embodiment of the present invention are shown. Chl a / b represent chlorophyll a and b, respectively; Carotenoids represents the total carotenoids in the biomass. All measurements were repeated three times. DETAILED DESCRIPTION

[0022] The present invention is described in detail below. It should be understood that the following description is only used to illustrate the present invention by way of example and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims. In addition, those skilled in the art understand that the technical solution of the present invention can be modified without departing from the spirit and purpose of the present invention.

[0023] As described above, the present inventors provide a large-scale method for efficiently treating saline wastewater by semi-continuously culturing Dunaliella salina strain and a microbial community comprising the algae strain and its symbiotic bacteria in saline wastewater.

[0024] In a first aspect, a method for treating saline wastewater is provided, comprising: semi-continuously culturing halophilic microorganisms in the saline wastewater; wherein the halophilic microorganisms are Dunaliella salina such as Dunaliella salina or a microbial community including the Dunaliella salina; and wherein the Dunaliella salina is an algae strain preserved on October 14, 2020 at the Guangdong Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with a preservation number of GDMCC No: 61230.

[0025] In a specific embodiment, the method further comprises: removing 40% to 60% of the volume of the treated saline wastewater from the culture system every day, and supplementing the same volume of the saline wastewater to be treated therein.

[0026] In a preferred embodiment, the method further comprises: removing 45% to 55% of the volume of the treated saline wastewater from the culture system every day, and supplementing the same volume of the saline wastewater to be treated therein.

[0027] In a more preferred embodiment, the method further comprises: removing 50% of the volume of the treated saline wastewater from the culture system every day, and supplementing the same volume of the saline wastewater to be treated therewith. The inventors of the present application have found that removing 50% of the volume of the treated saline wastewater from the culture system every day, and supplementing the saline wastewater to be treated therewith, so that the hydraulic retention time of the semi-continuous culture method is 2 days, can achieve the optimal wastewater treatment efficiency.

[0028] As used herein, the term "high salinity" refers to a salinity of more than 5% as known to those skilled in the art. In the context of the present invention, the term "salinity" is a scale known in the art to measure the salt content of seawater, and in the context of the present invention, it means the total number of grams of solid matter contained in each thousand (hundred) grams of saline wastewater after carbonates are converted into oxides, bromine and iodine are replaced by equivalent amounts of chlorine, and all organic matter is oxidized.

[0029] In a specific embodiment, the salt-containing wastewater includes processing water from the food industry, chemical industry, agriculture, textile industry, leather industry, etc., but is not limited thereto.

[0030] In a more specific embodiment, the saline wastewater is water for processing kelp. The preservation of kelp requires the use of a large amount of salt (mainly sodium chloride) to extend the shelf life of kelp, so in the rinsing process of kelp, a large amount of high-salinity processing water will be produced. Since the kelp undergoes high-temperature treatment during the rinsing process, this causes the high-salinity processing water to also contain a large amount of organic matter (mainly sodium alginate) and inorganic nutrients (mainly phosphates and nitrogen salts). If the high-salinity processing water containing a large amount of organic pollutants and inorganic nutrients is not effectively treated, it will cause great harm to the ecological environment of the water body, such as eutrophication of water bodies.

[0031] In a preferred embodiment, the saline wastewater initially has or is adjusted to have a salinity of 5% to 15%. In a more preferred embodiment, the saline wastewater initially has or is adjusted to have a salinity of 10% to 13%. In a most preferred embodiment, the saline wastewater initially has or is adjusted to have a salinity of 10%. The method for adjusting the salinity is not limited, for example, by diluting with water, or by adding sodium chloride. Those skilled in the art can make a choice according to the actual situation of the saline wastewater.

[0032] The inventors of the present application have found that under the reaction conditions of the present invention, Dunaliella salina strains cannot directly utilize organic carbon such as sodium alginate in saline wastewater as a carbon source for mixotrophic reproduction, and their symbiotic bacteria cannot utilize and degrade sodium alginate to convert it into an inorganic carbon source for use by microalgae. Therefore, in the treatment of saline wastewater such as kelp processing water, organic matter such as alginate in the wastewater can be removed before cultivation.

[0033] Therefore, in yet another specific embodiment, the method further comprises: a step of recovering organic matter in the saline wastewater before the semi-continuous culturing.

[0034] In a further specific embodiment, the organic matter may be sodium alginate. The recovered sodium alginate may be used as a potential and sustainable raw material for food, biopharmaceuticals and bioplastics in the fields of packaging, papermaking, textiles and wound dressings (Rinaudo, M. Biomaterials based on a natural polysaccharide: alginate. 2014 TIP, Volume 17, Issue 1, Pages 92-96, ISSN 1405-888X, https: / / doi.org / 10.1016 / S1405-888X(14)70322-5, incorporated herein by reference), but is not limited thereto.

[0035] In a further specific embodiment, the recovery can be carried out by calcium ion precipitation. If calcium ion is used to precipitate alginate, calcium alginate fiber can be formed by wet spinning, which has good tensile strength and can be used as a good cloth. Blended yarns and textiles of calcium alginate fiber have good feel and strength. Calcium alginate fiber is also a natural flame retardant material (Rinaudo, M. Biomaterials based on a natural polysaccharide: alginate. 2014 TIP, Volume 17, Issue 1, Pages 92-96, ISSN 1405-888X, https: / / doi.org / 10.1016 / S1405-888X (14) 70322-5, incorporated herein by reference). Therefore, the recovery of organic matter such as alginate can partially offset the cost of the entire wastewater treatment process, making wastewater treatment cost-effective.

[0036] In a specific embodiment, the saline wastewater comprises inorganic nutrient salts.

[0037] In further specific embodiments, the inorganic nutrient salts include nitrogen salts and / or phosphorus salts.

[0038] In a further specific embodiment, the inorganic nutrient salts include:

[0039] - a nitrogen salt having a concentration of 12 to 36 mg / L, preferably 20 to 25 mg / L, calculated as nitrogen element;

[0040] and / or

[0041] - a phosphorus salt having a concentration of 1 to 4.5 mg / L, preferably 1.5 to 2.5 mg / L, calculated as elemental phosphorus.

[0042] In further specific embodiments, the nitrogen salt comprises ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen. In a preferred embodiment, the nitrogen salt is ammonia nitrogen, for example NH4 + .

[0043] In a further specific embodiment, the phosphate salt comprises phosphate and / or phosphite. In a preferred embodiment, the phosphate salt is phosphate, for example, PO4 3+ .

[0044] In yet another specific embodiment, the conditions under which the Dunaliella salina strain or the microbial community grows in the saline wastewater include:

[0045] -Light intensity is 100 μmol / m 2 / s to 300 μmol / m 2 / s;

[0046] - A photoperiod of 18h / 6h to 6h / 18h light / dark;

[0047] - a temperature between 15°C and 35°C; and

[0048] - pH set to 7.0 to 7.8.

[0049] In a preferred embodiment, the conditions for the growth of the Dunaliella salina strain or the microbial community in the saline wastewater include:

[0050] -Light intensity is 150 μmol / m 2 / s to 250 μmol / m 2 / s;

[0051] - A photoperiod of 16h / 8h to 8h / 16h light / dark;

[0052] - a temperature of 20°C to 30°C; and

[0053] - pH set to 7.3 to 7.7.

[0054] In a more preferred embodiment, the conditions for the growth of the Dunaliella salina strain or the microbial community in the saline wastewater include:

[0055] -Light intensity is 200±20μmol / m 2 / s;

[0056] - A photoperiod of 14h / 10h to 10h / 14h light / dark;

[0057] - Temperature is 25±1°C; and

[0058] - pH was set to 7.4 ± 0.1.

[0059] The illumination can be achieved by using a light source known in the art, such as a fluorescent tube, an LED lamp, etc., but not limited thereto.

[0060] The pH value in saline wastewater can be regulated by introducing a carbon source, CO2. The growth of Dunaliella salina strains uses CO2 as a carbon source. Therefore, the amount of CO2 in the culture system will inevitably decrease as the strains grow, causing the pH value of the culture system to increase as the strains grow. Once it is detected that the pH value of the culture system exceeds a preset upper threshold, CO2 is introduced into the system until the pH value reaches a preset lower threshold. In addition, the pH value of the culture system can also be balanced by adding Na2CO3 to the saline wastewater, but is not limited thereto.

[0061] In a further specific embodiment, the semi-continuous culture conditions further include: introducing air or CO2 into the saline wastewater.

[0062] In yet another specific embodiment, said microbial community further comprises symbiotic bacteria in symbiosis with said Dunaliella tertiolecta.

[0063] In a preferred embodiment, the symbiotic bacteria may be Halomonas sp. or Idiomarina sp., but are not limited thereto.

[0064] In a more preferred embodiment, the Halomonas is a Halomonas strain deposited on October 14, 2020 at Guangdong Microbiological Culture Collection Center, Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with the deposit number GDMCC No: 61229.

[0065] In another more preferred embodiment, the marine-derived bacteria is a strain preserved on October 14, 2020 in the Guangdong Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with the preservation number GDMCC No: 61228.

[0066] It should be pointed out first that a stable microbial colony is the key to successfully treating saline wastewater, because halophilic symbiotic bacteria such as Halomonas and Halobacterium can reduce the COD of sewage by degrading other organic matter in the wastewater. In the treatment of saline wastewater such as kelp processing water, a feasible solution to reduce total organic carbon (TOC) is to introduce more naturally selected halophilic bacteria or artificially select halophilic bacteria from a strain library to form a richer microbial colony. Therefore, it is preferred to use a microbial community comprising Dunaliella salina and its symbiotic bacteria, such as the microbial community described in the second aspect of the present invention, to treat saline wastewater.

[0067] In a second aspect, the present invention provides a method for producing edible salt, the method comprising:

[0068] (a) implementing the method described in the first aspect to obtain treated saline wastewater;

[0069] (b) evaporating the treated saline wastewater to obtain edible salt.

[0070] The evaporation may be performed by methods well known in the art, and is not limited thereto.

[0071] As can be seen from the above, by using the semi-continuous culture method of the present invention, the Dunaliella salina strain and the microbial community preferably comprising the Dunaliella salina strain can almost completely absorb the inorganic nutrients in the saline wastewater within 24 hours; and the organic matter such as sodium alginate in the saline wastewater can be recovered by precipitation before the start of culture using the method in the prior art. Thus, the treated saline wastewater obtained by implementing the method described in the first aspect contains only a small amount of impurities, especially in the treatment of kelp processing water, because it is food processing water, the impurities remaining after treatment, whether in terms of composition or content, will not cause any harm to the human body, which enables the production of high-value table salt by evaporation means well known in the art. Therefore, compared with traditional wastewater treatment methods, the microbial treatment technology of the present invention provides a new, large-scale, environmentally friendly, cost-effective and sustainable way to treat high-salinity wastewater.

[0072] Hereinafter, the present invention will be described in more detail in conjunction with exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be considered as intended to interpret the scope of the present invention.

[0073] Example

[0074] Example 1. Isolation, purification and identification of Dunaliella salina and symbiotic bacteria

[0075] Isolation and purification of Dunaliella salina: The salinity of the kelp processing water sampled from Haizhibao and brought back to Jacobs University Bremen was close to 20%. After adding twice the f / 2 (Guillard 1975) culture medium nutrient salt to the sampled processing water, it was placed in a temperature-controlled room at 21±1°C for cultivation. After one month of cultivation, the concentration of microalgae cells in the sample increased significantly compared to the initial colorless water sample, such as Figure 1 As shown in A. An integer volume of algae liquid is evenly spread on an agar plate supplemented with twice f / 2 nutrient salts and cultured until a single colony of Dunaliella salina microalgae grows on the plate, as shown in Figure 1 B. Single colonies of microalgae are picked and transferred to separate culture bottles to form pure cultures, such as Figure 1 C. Among them, Figure 1 The pure cultured algae strains numbered 2-9 in C were selected for the following experiments because of their darkest color. Before the experiment to optimize the optimal salinity, Dunaliella salina was pre-cultured in culture media with different salinities (5%, 10%, 13% and 15%, with salinity adjusted by adding sodium chloride), as shown in Figure 2. Figure 1 As shown in D.

[0076] Isolation and purification of symbiotic bacteria: The cultured algae solution was spread on a Marine Broth (MB) agar plate adjusted to 13% salinity. After two days of cultivation in a 30°C incubator, bacterial monoclonal colonies ( Figure 1 E). Pick out monoclonal colonies with different morphological characteristics and inoculate them into separate liquid MB medium with a salinity of 13% ( Figure 1 F), continue culturing in a 30°C incubator to prepare for bacterial DNA extraction.

[0077] Morphological observation of Dunaliella salina: Halophilic microalgae were fixed with Lugol's iodine solution, and microscopic photos of halophilic microalgae were taken using a Motic AE30 inverted microscope and a C-mount digital microscope camera (60x objective lens). Figure 2 As shown. Figure 2 It can be seen that the isolated unicellular microalgae have a double flagella structure, are mobile, and the cell morphology is usually pear-shaped, fusiform or spherical, which are typical morphological characteristics of Dunaliella salina. There is a cup-shaped chloroplast in the middle of the cell, surrounded by many starch granules and lipid droplets containing carotenoids. The typical characteristics of Dunaliella salina are a thin and transparent plasma membrane and the lack of a hard cell wall, which allow the cell to quickly adapt to environmental changes. Dunaliella salina usually presents an elongated cell morphology in a favorable environment, but the cells are usually spherical when under stress.

[0078] Gene sequence identification: Since the isolated microalgae have similar morphological characteristics, 3 microalgae were selected from 10 pure culture algae and transferred to a larger volume of culture medium to accumulate biomass for extraction of genomic DNA. The environmental conditions for microalgae culture are: light intensity 150μmol / m 2 / s, 21±1°C, and a light rhythm of 18 hours light / 6 hours dark. This experiment used a modified Johnson's medium (salinity 3.3%) to culture microalgae, as shown in Table 1 below.

[0079] Table 1. Modified Johnson's medium

[0080]

[0081] The microalgae biomass was harvested by centrifugation at 2000 g for 10 minutes and then freeze-dried overnight. The freeze-dried algae powder genomic DNA was extracted using a DNA extraction kit (NucleoSpin Plant II, Macherey Nagel, Germany). In order to achieve better extraction results, the cooled and dried algae powder was first ground in a mortar with liquid nitrogen. 4-10 mg of ground algae powder was collected in a 1.5 mL Eppendorf tube for DNA extraction. The extracted DNA concentration was measured using a spectrophotometer (BioSpectrometer, Eppendorf, Germany) and the quality of the extracted DNA was evaluated. The extracted DNA was amplified by polymerase chain reaction using a thermal cycler (T100 thermal cycler, Bio-Rad, USA), where: for microalgae, ITS was amplified using primer sequences ITS4 (SEQ ID NO: 4: 5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (SEQ ID NO: 5: 5'-TCCGTAGGTGAACCTGCGG-3'); for symbiotic bacteria, 16S rDNA was amplified using universal primer sequences 27F (SEQ ID NO: 6: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO: 7: 5'-GGTTACCTTGTTACGACTT-3'). The total PCR system was 50 μL, including: 0.6 μL forward primer and 0.6 μL reverse primer (10 mM), 0.6 μL dNTPs (10 mM), 0.6 μL Taq DNA polymerase, 0.5 μL 10×ThermoPol reaction buffer, 2 μL DNA template and 40.6 μL MillIQ water. The treatment group with deionized water as the template served as the negative control group. The PCR program included in sequence: pre-denaturation (95°C, 3 min), DNA denaturation (95°C 30 s, 30 cycles), annealing (60°C 30 s) and extension (72°C 2 min). The quality of the PCR amplification product was detected by 1% agarose gel electrophoresis before sequencing, and the results are shown in Figure 2. Figure 3 The PCR amplification product was then bidirectionally sequenced using forward and reverse primers from Eurofins. The sequencing results showed that the ITS sequence of the microalgae was SEQ ID NO: 1, and the 16S rDNA sequences of the symbiotic bacteria were SEQ ID NO: 2 (Halomonas) and SEQ ID NO: 3 (Halogenobacterium).

[0082] The sequence of SEQ ID NO:1 is as follows: TGTCTGGCCCACCAATTCTCTCCGGAGTTTTGGTGCTTCCTGGGCAGCATTAACTTGCTGTCTGGGTTGGGCTCGGCTGCCCAAATCCAATTTGGGTAGTCGGGTTGGTCCTTTACTAACCAACAACACCAAACAAAACCTAAAGCCAAAGATATGTGATCGGCCTAGCCGTCACATCCTAACTGAGACAACTCTCAACAACGGATATCTTGGCTCTCGCAACGATGAAGAACGCAGCGAAATGCGATACGTAGTGTGAATTGCAGAATTCCGTGAATCATCAAATCTTTGAACGCAAATTGCGTCCGCGGCTTCGGCCAAGGGCATGTTTGCCTCAGCGTCGGGTTAATCTCACTAACCACTCCCCCATGCGAGGAGTACTGTTAGTGGACCTGGCTGTTCCAGAGCTTGGGTGCTTCATTGCCCCAGCAAAGGCTCTGGATCAGCTGAAGAGCAGAGGCTAGCTCAAGGACCCGTCAAGGGCCGCAACTGGGTAGGCAGCTTTCGCTCGCTATTTCTAGTTGTTGGCTTGGGACCATGGGCTTGGCCCTCAAACAGGAACCCTTCTTTTTTCTCGACCTGAGCTCAAGCAAGACCACCCGCT。

[0083]

[0084] The sequence of SEQ ID NO:3 is as follows: GCGGTAACAGAGAGAAGCTTGCTTCTCTGCTGACGAGCGGCGGACGGGTGAGTAATACTTGGGAATTTGCCTTTAGGCGGGGGACAACCACGGGAAACTGTGGCTAATACCGCATAATGTCTACGGACCAAAGTGGGGGACCTTCGGGCCTCACACCTAAAGATGAGCCCAAGCGGGATTAGCTAGTTGGTGGGGTAAAGGCTCACCAAGGCGACGATCCCTAGCTGTTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGACGAGGAAGGGTGTTGTGTTAATAGCACAGCACATTGACGTTAGTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGTAGGCGGCGTGTTAAGCAAGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTTGAACTGGCATGCTAGAGTCCTGAAGAGGGTGGTAGAATTTCCAGTGTAGCGGTGAA.

[0085] The above DNA sequencing results were uploaded to the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the basic local sequence alignment tool (BLAST) function was used for sequence alignment.

[0086] Based on the above morphological and molecular biological information, the isolated microorganisms were identified as Dunaliella sp., Halomonas sp. and Idiomarina sp.

[0087] Example 2: Batch culture of Dunaliella salina in universal culture medium at different salinities

[0088] Although Dunaliella salina has the characteristic of tolerating high salinity, salinity may affect the wastewater treatment efficiency and the accumulation of carotenoids. Therefore, before using Dunaliella salina to treat process water, it is necessary to optimize the salinity of the process water. To this end, in this embodiment, the optimal salinity suitable for Dunaliella salina to treat process water was tested.

[0089] The culture conditions are as follows: 4 times f / 2 universal medium was used to culture Dunaliella salina, where: the initial OD 725 The value was about 0.2; the salinity of the culture medium was adjusted by adding sodium chloride; the lighting was provided by fluorescent tubes: the light intensity was 200 μmol / m 2 / s; the photoperiod was 14 h light / 10 h dark; the room temperature was maintained at 25 ± 1 °C; the pH was set to 7.4 with a hysteresis effect of 0.1 (adjusted by the CO2 valve, opened when the pH value reached 7.5; closed when the pH value was lower than 7.3). During the culture period, the OD of the algae solution was measured every day. 725 The value was used to monitor the growth curve of the microalgae and the specific growth rate was calculated using the following formula:

[0090] μ=(ln X t -ln X0) / (d-d0)

[0091] Where μ represents the specific growth rate, X t X0 and X1 represent the OD at time point d and time point d0, respectively. 725 value.

[0092] Two batches of cultivation were carried out in the universal medium to obtain stable growth results. The salinity of the first cultivation was 3.5%, 5%, 10% and 15%, respectively. Figure 4 As shown in A. It can be found that Dunaliella salina grown in culture medium with salinity of 3.5% and 5% grows faster than Dunaliella salina grown in culture medium with salinity of 10% and 15%. Dunaliella salina grown in culture medium with salinity of 10% is inhibited in the early growth stage and enters the exponential growth phase after the second day, which indicates that Dunaliella salina needs 48 hours to adapt to the high salinity environment. By the 8th day of culture, the OD of Dunaliella salina grown in culture medium with salinity of 10% was 2.3477 W / m. 725 Values ​​and OD of Dunaliella salina grown at salinity of 3.5% and 5% 725 The values ​​are basically the same. However, Dunaliella salina inoculated into a culture medium with a salinity of 15% completely stopped growing on the first day of culture, and on the second day of culture, the cells turned white and lost their complete cell structure and chromatin.

[0093] In the second batch of culture, Dunaliella salina that had adapted to 10% salinity was used as inoculum to be inoculated into culture media with salinities of 10%, 13%, and 15%. The growth curves were as follows: Figure 4 B. Compared with the first batch of cultures, Dunaliella salina entered the exponential growth phase within 24 hours, and Dunaliella salina grown in 10% salinity reached the plateau phase on the sixth day of culture. Although the second batch of Dunaliella salina was able to grow in the culture medium with a salinity of 15%, Figure 4 B shows that its growth is still significantly inhibited. Figure 4 The specific growth rates of Dunaliella tertiolecta in the exponential growth phase in all groups of the two batch cultures are shown in C. Dunaliella tertiolecta adapted to a high salinity environment exhibited the highest specific growth rates in the culture medium at 10% and 13% salinity.

[0094] Example 3: Cultivation of Dunaliella salina in synthetic process water

[0095] According to the analysis report of the original saturated process water provided by Haizhibao Company, synthetic process water was prepared to simulate the actual wastewater treatment situation. According to the information provided by Haizhibao Company, the source of the process water is seawater. Therefore, in this experiment, in order to better simulate the actual water source that needs to be treated, synthetic process water was prepared in the laboratory using North Sea seawater, and the salinity of the seawater was increased by adding sodium chloride, and NH4Cl and KH2PO4 were added as inorganic nutrients. The nutrient and total organic carbon (TOC) contents of the prepared process water with salinities of 5%, 10% and 15% are shown in Table 2 below. Of course, it can be understood that in order to achieve the maximum nutrient recovery efficiency and biomass yield, the saturated process water can be diluted with low-salinity process water in the workshop to achieve the optimal salinity. Here, we assume that the low-salinity process water does not contain nutrients and organic pollutants.

[0096] Table 2: Nutrient and organic carbon content in process water of different salinities after mixing

[0097]

[0098] *Assuming that TOC is derived from sodium alginate

[0099] Dunaliella salina was pre-cultured to the late exponential growth phase before inoculation. During the culture period, the OD of the algae solution was measured every day. 725 The value is used to monitor the growth of algae species, and the remaining nutrient concentration in the algae solution is collected and measured every two days. + -N) and active phosphate (PO4 3+ -P) were measured using the standards ISO 11732-E23:2005-05 and ISO6878-D11:2004-09. The results are as follows Figure 5 shown.

[0100] like Figure 5As shown in A, Dunaliella salina grew rapidly in synthetic process water with salinity of 5% and 10%, and entered the plateau phase on the second and third days of culture, respectively, while it grew slowly in synthetic process water with salinity of 15%, but continued to grow during the 6 days of culture. In other words, although the process water with salinity of 15% contained a higher nutrient concentration, the growth of Dunaliella salina was slower than that of the experimental groups with salinity of 5% and 10%.

[0101] In all experimental groups, the growth of Dunaliella salina and the concentration of residual nutrients in the process water showed a good correlation, e.g. Figure 5 B and Figure 5 C. The absorption of ammonia nitrogen and active phosphate is almost synchronous, which indicates that the ratio of nitrogen and phosphorus in the process water is very favorable for the growth of Dunaliella salina. Figure 5 As shown in Figure C, Dunaliella salina can recover almost all the nutrients in the process water with salinity of 5% and 10% within two days after the start of cultivation, but it takes 6 days to completely absorb the nutrients in the process water with a salinity of 15%. In addition, compared with other experimental groups, Dunaliella salina showed the highest removal efficiency of ammonia nitrogen and active phosphate (calculated as phosphorus element therein) in the process water with a salinity of 10%, which were 10.17 mg / L / d and 1.25 mg / L / d, respectively. Even though the specific growth rate of Dunaliella salina in 5% salinity (0.42) was slightly higher than the specific growth rate in 10% salinity (0.36), Dunaliella salina showed the highest nutrient absorption efficiency in the process water with a salinity of 10%. It can be seen that 10% salinity is the optimal salinity for Dunaliella salina to treat saline wastewater, and the hydraulic retention time for treating this process water by Dunaliella salina is two days.

[0102] Example 4: Batch and semi-continuous cultivation of Dunaliella salina in bag bioreactors

[0103] In this example, the volume of process water to be treated was expanded in a larger suspended bag bioreactor (Phytobags from iSeaMC) to demonstrate the feasibility of large-scale treatment of saline wastewater using the Dunaliella salina or microbial community of the present invention.

[0104] In addition, in order to further increase the added value of the harvested biomass, strong light and nutrient starvation stress were used during the semi-continuous culture of this example to induce Dunaliella salina to synthesize high-value carotenoids.

[0105] Batch culture:

[0106] First, Dunaliella salina was cultured in a bottle bioreactor with 1L of process water to accumulate biomass. After saturated process water was prepared, the salinity was diluted to 10% with tap water. Two vertical bag reactors were prepared and placed in a temperature-controlled room in the greenhouse. When Dunaliella salina grew to the stationary stage, the Dunaliella salina that had been adapted to 10% salinity in advance was transferred to the bag bioreactor, and 1L of process water and 2L of nutrient salts were added to make the total volume reach 2L. Three days later, 0.5L of process water and 2.5L of nutrient salts were added to make the total volume reach 2.5L. When the microalgae grew to the stationary stage again, 7.5L of process water and 10L of nutrient salts (NH4Cl, KH2PO4, Na2CO3 and vitamins) were added to make the total volume reach 10L. Sodium alginate (8.89g) (Alginate+) was added to one of the reactors. Illumination was provided by fluorescent tubes: the light intensity was 200μmol / m 2 / s; the photoperiod was 14 h light / 10 h dark; the room temperature was maintained at 25±1°C; the pH was set to 7.7, and the delay was 0.1 (adjusted by the CO2 valve, opened when the pH value reached 7.8; closed when the pH value was lower than 7.6). Environmental factors remained constant throughout the experiment.

[0107] Measure the OD of the algae solution every day 725 The values ​​and biomass concentrations were obtained by batch culture for a total of 7 days. When measuring the biomass concentration, 15 mL of algae solution was filtered onto a 0.7 μm glass fiber filter using a vacuum pump. The filter membrane was washed with deionized water and dried overnight in a 60°C oven before use. The dried filter membrane was weighed using a fine balance. After filtering the algae solution onto the filter membrane, the filter membrane was rinsed three times with 5 mL of deionized water to remove residual salts and alginate, and the filter membrane was placed in an oven at 60°C to dry overnight. The dried filter membrane was weighed again using a fine balance. The biomass concentration in the algae solution was calculated based on the change in filter membrane mass and the volume of filtered algae solution. All measurements were repeated three times to ensure the accuracy of the measurement data.

[0108] Semi-continuous culture:

[0109] On the 7th day of culture, according to OD 725The value confirms that the microalgae has entered the plateau phase. 3.3L of algae liquid without sodium alginate was thoroughly mixed and added to 6.7L of synthetic process water with a salinity of 10%, and 10L of equivalent nutrients (NH4Cl, KH2PO4, Na2CO3 and vitamins) were added. Semi-continuous culture uses exactly the same environmental conditions as batch culture. According to previous experimental results in bottle reactors, Dunaliella salina can completely recover nutrients within two days, so semi-continuous culture started on the second day. For the next three days, 5L of algae liquid was harvested every day, and then 5L of synthetic process water with a salinity of 10% to be treated was added. Samples were taken every day before replacing the process water to measure the concentrations of remaining ammonia nitrogen and active phosphoric acid (in terms of phosphorus element) in the process water, as described above, to evaluate the effect of wastewater treatment.

[0110] Carotenoid synthesis induction and measurement:

[0111] On the fifth day of batch culture, strong light stress (LED light provided 700 μmol / m 2 / s to 1000 μmol / m 2 / s) and nutrient starvation stress (no fresh process water was added). During the semi-continuous culture period, the OD of the harvested algae solution was measured every day. 725 , biomass concentration, and light and pigment concentrations. All measurements were repeated three times.

[0112] 30-50 mL of algae liquid was collected by centrifugation (3000 g, 10 min) every day. After discarding the supernatant, the biomass was stored at -20 °C, and three replicates were set. All samples needed to be freeze-dried for 4-8 hours until completely dry before pigment analysis. Photosynthetic pigments were extracted using 100% acetone under weak light conditions. A magnetic stirrer was used to stir for 30 min during the extraction process. After the extraction process was completed, the mixture was centrifuged at 3000 g for 10 min, and the organic phase was diluted 2-4 times. The absorbance of the sample at wavelengths of 645 nm, 662 nm and 470 nm was measured by a spectrophotometer. The pigment concentration was calculated by the following formula (see Srinivasan, R., Mageswari, A., Subramanian, P. et al. Bicarbonate supplementation enhances growth and biochemical composition of Dunaliella salina V-101 by reducing oxidative stress induced during macronutrient deficit conditions. Sci Rep 8, 6972 (2018), https: / / doi.org / 10.1038 / s41598-018-25417-5, incorporated herein by reference):

[0113] Chlorophyll a (chl a) (μg / mL) = 11.75 (A 662 )–2.35(A 645 )

[0114] Chlorophyll b (chl b) (μg / mL) = 18.61 (A 645 )–3.96(A 662 )

[0115] Total carotenoids (μg / mL) = (1000A 470 –2.270Chl a–81.4Chl b) / 198

[0116] Figure 6 The results of treating synthetic process water by batch culture of Dunaliella salina in a bag bioreactor are shown, including: (A) comparison of algal liquid color between Alginate- and Alginate+ groups; (B) growth curve; (C) biomass accumulation. Figure 6As shown, Dunaliella salina showed very similar growth curves and biomass accumulation levels in these two different processing waters. Dunaliella salina entered the plateau phase on the third day of culture in both processing waters, but the biomass concentration continued to increase until the sixth day, with the maximum biomass concentrations in the Alginate- and Alginate+ experimental groups being 0.3 g / L and 0.33 g / L, respectively. Dunaliella salina had the largest specific growth rate between the first and second days in both the Alginate- and Alginate+ experimental groups, which were 0.52 and 0.49, respectively, representing that the doubling time of the microalgae cells was 1.32 and 1.47 days, respectively. The above results all show that sodium alginate has no significant effect on the growth and biomass accumulation of Dunaliella salina. The possible reason for this result is that Dunaliella salina cannot directly use sodium alginate as a carbon source for mixotrophic reproduction, and its symbiotic bacteria cannot use and degrade sodium alginate to convert it into an inorganic carbon source for microalgae to use. It can be seen that the results in the bag-shaped bioreactor of this example are basically consistent with the results in the bottle-shaped reactor of Example 3, showing good repeatability.

[0117] Figure 7 The figure shows the changes in the concentration of photosynthetic pigments of Dunaliella salina during batch culture, where Alginate+ / - represents the presence / absence of sodium alginate in the processing water, Chl a / b and carotenoids represent chlorophyll a / b and total carotenoids, respectively. Figure 7 It can be seen that although the concentrations of different photosynthetic pigments in different experimental groups are different, the change trends in the bag bioreactor culture are basically the same, all reaching the maximum on the fourth day of culture, and then the concentration begins to decrease. Comparing the results of the experimental groups with and without alginate, it can be seen that on the fourth day of culture, the concentration of all photosynthetic pigments in the experimental group without alginate was significantly higher than that in the experimental group with alginate (p<0.05). The highest concentrations of carotenoids synthesized by Dunaliella salina in the process water without and with alginate reached 2.05 mg / L and 1.86 mg / L, respectively. According to the carotenoid concentration and biomass concentration, the proportion of carotenoids in cell dry weight in Alginate- and Alginate+ reached 0.78% and 0.70%, respectively.

[0118] From the above experimental results, it can be seen that the presence of sodium alginate during the culture process is not conducive to the synthesis of carotenoids by Dunaliella salina, so the pretreatment of recovering alginate before the culture of Dunaliella salina is conducive to the synthesis of carotenoids. At the same time, it can also be seen that although the biomass concentration of Dunaliella salina continues to grow after entering the plateau period, the total carotenoid concentration in the algae solution decreases instead.

[0119] Figure 8Results are shown for semi-continuous cultivation of Dunaliella tertiolecta in a bag reactor treating synthetic process water. Figure 8 A shows the change in color of the Dunaliella salina algae solution during the entire culture process. It can be seen with the naked eye that the color of the algae solution becomes darker and darker over time. Figure 8 B and Figure 8 C shows the OD of the algae solution measured every day. 725 and biomass concentration (dry weight). Figure 8 B shows that each time the algae liquid is harvested and fresh processing water is added, the OD 725 The value can be quickly restored within 24 hours. Between the second and fifth days of continuous addition of fresh process water, the average daily specific growth rate can reach 0.6, which means that the average cell doubling time is 1.16 days.

[0120] Fig. 9 The residual nutrient concentration in the synthetic process water during the semi-continuous culture is shown. Fig. 9 It can be seen that Dunaliella salina can completely remove the active phosphate in the process water on the second day, so that the remaining active phosphate concentration is lower than the limit of the test method (less than 0.1 mg / L). Similarly, the ammonia nitrogen concentration also dropped significantly to 6 mg / L on the second day. At the same time, it can be seen that the ammonia nitrogen and active phosphate in the process water replaced daily are completely absorbed within 24 hours (before the next replacement), without causing the accumulation of residual nutrients. The removal efficiency of ammonia nitrogen and active phosphate (calculated as phosphorus element) is as high as 11.14 mg / L / d and 1.35 mg / L / d, respectively.

[0121] It should be pointed out that starting from the second day of cultivation, the processing water after 24 hours of treatment can reach the level of Class I water in the current comprehensive sewage discharge standard. According to the standard of GB8978-1996, the concentration of active phosphate (calculated as phosphorus element) in the discharged sewage is required to be less than 0.5 mg / L, and the ammonia nitrogen is less than 15 mg / L. These experimental results prove that under the condition of a hydraulic retention time of two days, Dunaliella salina can continuously and effectively treat 10% salinity kelp processing water, and the treated processing water meets the relevant indicators in the Class I discharge standard, as shown in Table 3 below.

[0122] Table 3: Some key indicators in the comprehensive sewage discharge standard GB8978-1996

[0123]

[0124] Figure 8 B and Figure 8 C also shows the OD of Dunaliella salina after stress in batch culture and semi-continuous culture. 725As can be seen from the figure, after 24 hours of strong light stress, that is, on the sixth day, the OD 725 However, if the strong light stress time is extended (from the sixth to the ninth day), the OD 725 Similarly, the biomass (dry weight) concentration of the algae solution also increased rapidly during the 24-hour strong light stress period, but the biomass concentration remained basically constant from the sixth to the ninth day.

[0125] Fig.10 The figure shows the changes in the concentration of some photosynthetic pigments (chlorophyll a / b and total carotenoids) during the semi-continuous culture. As can be seen from the figure, after 24 hours of high light stress, that is, on the sixth day of semi-continuous culture, the concentration of chlorophyll a / b began to decrease, but the total carotenoid concentration reached a maximum value (2 mg / L) on the sixth day. The carotenoid content in Dunaliella salina reached 0.57% of dry weight on the sixth day. However, further extending the light time (sixth to ninth day) will lead to a decrease in the concentration of carotenoids. Sequence Listing <110> Shandong Haizhibao Marine Technology Co., Ltd. <120> Methods for treating saline wastewater <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 604 <212> DNA <213> Dunaliella salina <400> 1 tgtctggccc accaattctc tccggagttt tggtgcttcc tgggcagcat taacttgctg 60 tctgggttgg gctcggctgc ccaaatccaa tttgggtagt cgggttggtc ctttaactaac 120 caacaacacc aaacaaaacc taaagccaaa gatatgtgat cggcctagcc gtcacatcct 180 aactgagaca actctcaaca acggatatct tggctctcgc aacgatgaag aacgcagcga 240 aatgcgatac gtagtgtgaa ttgcagaatt ccgtgaatca tcaaatcttt gaacgcaaat 300 tgcgtccgcg gcttcggcca agggcatgtt tgcctcagcg tcgggttaat ctcactaacc 360 actcccccat gcgaggagta ctgttagtgg acctggctgt tccagagctt gggtgcttca 420 ttgccccagc aaaggctctg gatcagctga agagcagagg ctagctcaag gacccgtcaa 480 gggccgcaac tgggtaggca gctttcgctc gctatttcta gttgttggct tgggaccatg 540 ggcttggccc tcaaacagga acccttcttt tttctcgacc tgagctcaag caagaccacc 600 cgct 604 <210> 2 <211> 1303 <212> DNA <213> Halomonas sp. <400> 2 aacaggtcca gcttgctgga cgctgacgag cggcggacgg gtgagtaacg cataggaatc 60 tacccggtag tgggggataa cctggggaaa cccaggctaa taccgcatac gtcctacggg 120 agaaaggggg cttaggctcc cgctattgga tgagcctatg ccggattagc tggttggtga 180 ggtaatggct caccaaggcg acgatccgta gctggtctga gaggatgatc agccacatcg 240 ggactgagac acggcccgaa ctcctacggg aggcagcagt ggggaatatt ggacaatggg 300 ggcaaccctg atccagccat gccgcgtgtg tgaagaaggc cctcgggttg taaagcactt 360 tcagcgagga agaacgcctg tcggttaata cccggcagga aagacatcac tcgcagaaga 420 agcaccggct aactccgtgc cagcagccgc ggtaatacgg agggtgcaag cgttaatcgg 480 aattactggg cgtaaagcgc gcgtaggtgg cttgataagc cggttgtgaa agccccgggc 540 tcaacctggg aacggcatcc ggaactatga ggctagagtg caggagagga aggtagaatt 600 cccggtgtag cggtgaaatg cgtagagatc gggaggaata ccagtggcga aggcggcctt 660 ctggactgac actgacactg aggtgcgaaa gcgtgggtag caaacaggat tagataccct 720 ggtagtccac gccgtaaacg atgtccaccc accctttggt tccttagccc acttggtggc 780 gaagttaccc caataagtgg cccccttggg aagtccgccc ccaggtttaa acttcaaaga 840 aattgcgggg ggccccccca agcgttggac caggtggttt atttgaatcc accgggaaga 900 cccttccttc cccttgcctt cttgggactt ggtaagaaat ccctgggtcc tttgggaagg 960 ccagagccag gtgttccagg ccgttgtcca cttgggttgg ggaaatgtgg ggttaattcc 1020 ggtacgaagc ccacccctgt cctttttttc ccaccgattc ggttgggaac tctagggaga 1080 ctgccggtga caaaccggag gaaggtgggg acgacgtcaa gtcatcatgg cccttacggg 1140 tagggctaca cacgtgctac aatggccggt acaaagggcg gcgagctcgc gagagtcagc 1200 gaatccctta aagccggtct cagtccggat cggagtctgc aactcgactc cgtgaagtcg 1260 gaatcgctag taatcgtgca tcagaatggc acggtgaata cgt 1303 <210> 3 <211> 1322 <212> DNA <213> Idiomarina sp. <400> 3 gcggtaacag agagaagctt gcttctctgc tgacgagcgg cggacgggtg agtaatactt 60 gggaatttgc ctttaggcgg gggacaacca cgggaaactg tggctaatac cgcataatgt 120 ctacggacca aagtggggga ccttcgggcc tcacacctaa agatgagccc aagcgggatt 180 agctagttgg tggggtaaag gctcaccaag gcgacgatcc ctagctgttc tgagaggatg 240 atcagccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 300 attgcacaat gggggaaacc ctgatgcagc catgccgcgt gtgtgaagaa ggccttcggg 360 ttgtaaagca ctttcagcga cgaggaaggg tgttgtgtta atagcacagc acattgacgt 420 tagtcgcaga agaagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc 480 aagcgttaat cggaattact gggcgtaaag cgtacgtagg cggcgtgtta agcaagatgt 540 gaaagccccg ggctcaacct gggaattgca ttttgaactg gcatgctaga gtcctgaaga 600 gggtggtaga atttccagtg tagcggtgaa atgcgtagat attggaagga ataccggtgg 660 cgaaggcggc cacctggtca gagactgacg ctgaggtacg aaagcgtggg gagcaaacag 720 gattagatac cctggtagtc cacgccgtaa acgatgtcaa ctagttgttc gtgtcattaa 780 gacgtgagta acgcagctaa cgcactaagt tgaccgcctg gggagtacgg ccgcaaggtt 840 aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat 900 gcaacgcgaa gaaccttacc atcccttgac atccagtgaa ttttccagag atggattagt 960 gccttcggga acactgagac aggtgctgca tggctgtcgt cagctcgtgt tgtgagatgt 1020 tgggttaagt cccgcaacga gcgcaaccct tatccttagt tgccagcacg taatggtggg 1080 aactctgggg agactgccgg tgataaaccg gaggaaggtg gggacgacgt caagtcatca 1140 tggcccttac gggatgggct acacacgtgc tacaatggcg catacaaagg gcagcaagcc 1200 agcgatggtt agcgaatctc ataaagtgcg tcgtagtccg gattggagtc tgcaactcga 1260 ctccatgaag tcggaatcgc tagtaatcgt ggatcagaat gccacggtga atacgttccc 1320 gg 1322 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 tcctccgctt attgatatgc 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <400> 5 tccgtaggtg aacctgcgg 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 agagtttgat cctggctcag 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 ggttaccttg ttacgactt 19

Claims

1. A method for treating saline wastewater, comprising: Semi-continuously culturing halophilic microorganisms in the saline wastewater; wherein the saline wastewater is water for kelp processing; wherein the halophilic microorganism is Dunaliella sp. or a microbial community including the Dunaliella sp.; and wherein the Dunaliella sp. is an algae strain preserved in Guangdong Microbiological Culture Collection Center on October 14, 2020, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, with a preservation number of GDMCC No: 61230; Wherein, the kelp processing water includes inorganic nutrient salts, and the inorganic nutrient salts include: - ammonia nitrogen in a concentration of 12 to 36 mg / L calculated as elemental nitrogen; and / or - a phosphorus salt having a concentration of 1 to 4.5 mg / L calculated as elemental phosphorus, the phosphorus salt being selected from phosphates and / or phosphites; The kelp processing water initially has or is adjusted to have a salinity of 5% to 15%.

2. The method according to claim 1, wherein: The method further comprises: removing 40% to 60% of the volume of the treated saline wastewater from the culture system every day, and supplementing the same volume of the saline wastewater to be treated therein.

3. The method according to claim 2, wherein: The method further comprises: removing 45% to 55% of the volume of the treated saline wastewater from the culture system every day, and supplementing the same volume of the saline wastewater to be treated therein.

4. The method according to any one of claims 1 to 3, wherein: The saline wastewater initially has or is adjusted to have a salinity of 10% to 13%.

5. The method according to any one of claims 1 to 3, wherein: The saline wastewater initially has or is adjusted to have a salinity of 10%.

6. The method according to any one of claims 1 to 3, wherein: The method further comprises: a step of recovering organic matter in the saline wastewater before the semi-continuous culturing.

7. The method according to claim 6, wherein: The organic matter is sodium alginate, and the recovery is carried out by a calcium ion precipitation method.

8. The method according to any one of claims 1 to 3, wherein: The inorganic nutrient salts include: - Ammonia nitrogen NH4 with a concentration of 20 to 25 mg / L as nitrogen element + ; and / or - Phosphate PO4 with a concentration of 1.5 to 2.5 mg / L as elemental phosphorus 3+ .

9. The method according to any one of claims 1 to 3, wherein: The conditions of the semi-continuous culture include: a. Light intensity is 100 μmol / m 2 / s to 300 μmol / m 2 / s; b. The photoperiod is 18 hours / 6 hours to 6 hours / 18 hours light / dark; c. Temperature is between 15°C and 35°C; d. pH is 7.0-7.

8.

10. The method according to claim 9, wherein: The light intensity was 150 μmol / m 2 / s to 250 μmol / m 2 / s.

11. The method according to claim 9, wherein: Light intensity: 200±20μmol / m 2 / s.

12. The method according to claim 9, wherein: The photoperiod was 14 h / 10 h to 10 h / 14 h light / dark.

13. The method according to claim 9, wherein: The temperature is 20℃ to 30℃.

14. The method according to claim 9, wherein: The temperature is 25±1℃.

15. The method according to claim 9, wherein: pH is 7.3-7.

7.

16. The method according to claim 9, wherein: The pH was 7.4 ± 0.

1.

17. The method according to any one of claims 1 to 3, wherein: The semi-continuous culture conditions also include: introducing air or CO2 into the saline wastewater.

18. The method according to any one of claims 1 to 3, wherein: The microbial community also includes symbiotic bacteria that coexist with the Dunaliella salina, and the symbiotic bacteria are Halomonas sp. and Idiomarin sp.

19. The method according to claim 18, wherein: The Halomonas is a Halomonas strain deposited on October 14, 2020 at Guangdong Microbiological Culture Collection Center, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, with a deposit number of GDMCC No: 61229; and The Haiyuan bacteria is a strain preserved on October 14, 2020 in the Guangdong Provincial Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with the preservation number GDMCC No: 61228.

20. A method for producing edible salt, the method comprising: (a) implementing the method according to any one of claims 1 to 19 to obtain treated saline wastewater; (b) evaporating the treated saline wastewater to obtain edible salt.

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