CN116042457B - Sea bacillus with denitrification and nitrate dissimilation reduction functions as well as culture method and application thereof - Google Patents
Sea bacillus with denitrification and nitrate dissimilation reduction functions as well as culture method and application thereof Download PDFInfo
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- 229910002651 NO3 Inorganic materials 0.000 title claims abstract description 29
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 28
- 230000009467 reduction Effects 0.000 title claims abstract description 17
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000206597 Marinobacter hydrocarbonoclasticus Species 0.000 description 1
- 241000337866 Marinobacter sp. SL013A-34A-1 Species 0.000 description 1
- 241001169494 Marinobacter sp. sw0106-07 Species 0.000 description 1
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000206744 Phaeodactylum tricornutum Species 0.000 description 1
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- 239000008272 agar Substances 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
- C02F3/325—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae as symbiotic combination of algae and bacteria
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- C02F2101/105—Phosphorus compounds
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Abstract
The invention discloses a sea bacillus with denitrification and nitrate dissimilation reduction into ammonium functions, a culture method and application thereof, and belongs to the technical field of environmental biology. The preservation number of the sea bacillus (Marinobacter sp.) is CGMCC No.25998. Experiments prove that the method can perform DNRA under the condition of low C/N, preferentially perform DNF under the condition of high C/N, and realize the effective recycling of nitrogen and phosphorus in the seawater culture tail water with low C/N and high concentration nitrate by symbiotic of the marine bacilli and microalgae, and can realize the standard discharge of the seawater culture tail water in a short time.
Description
Technical Field
The invention relates to the technical field of environmental biology, in particular to a sea bacillus with denitrification and nitrate dissimilation reduction into ammonium functions, a culture method and application.
Background
China is the largest mariculture country in the world, and the mariculture industry has become one of the pillar industries of national economy. Compared with the traditional culture mode, the seawater circulating water culture has the advantages of water and land saving, high unit yield and controllable environment, and is an important direction of green development of the seawater culture industry. Nitrogen is the main subject of aquaculture tail water treatment. In the bait casting type intensive high-density cultivation mode, bait casting is a main source of nitrogen pollution, and is decomposed and converted into NH 4 + And NO 2 - Has strong toxicity to cultured organisms, and industrial circulating water culture is generally provided with a biological filter to convert the cultured organisms into NO with low biological toxicity through nitration reaction 3 - And 90-95% of the water body is recycled. Thus, "nitrate accumulation" is a common phenomenon in industrial circulating water systems, and untreated tail water discharge can lead to off-shore environmental pollution. At present, microbial denitrification is a conventional denitrification means, and a large amount of NO is in the long term 3 - -N by N 2 The release of this form into the atmosphere, while to some extent slowing off-shore nitrogen emissions, can result in significant loss of nitrogen resources. Therefore, the recycling of nitrogen in the mariculture tail water has important significance for the efficient green development of the mariculture industry.
At present, a micro-filter in a seawater land-based industrial circulating water culture system can remove most of solid particles, and a primary/secondary/tertiary biological filter can further intercept suspended particles and toxic and harmful NH 4 + And NO 2 - Conversion to less biotoxic NO 3 - Untreated tail water emissions can lead to offshore environmental pollution. In general, microbial denitrification is an effective means for denitrification of water, and unlike industrial wastewater and domestic sewage, the seawater circulating water culture tail water has the characteristics of low C/N, high salinity, low load and the like, so that the treatment difficulty is high, accurate external carbon source is required for efficient denitrification, and the carbon source quantity is insufficientThe denitrification rate is inhibited, and secondary pollution is easily caused by excessive carbon source quantity; on the other hand, the carbon source addition can greatly increase the water treatment cost and greatly compress the cultivation profit. Therefore, the resource utilization of the mariculture tail water is an effective measure facing the important national demands, and in recent years, the algae-bacteria symbiotic treatment technology combining the absorption of microalgae to nitrogen and phosphorus and other nutrient substances with the efficient degradation capability of bacteria is becoming a new hot spot for researching the field of the resource utilization of the tail water.
Disclosure of Invention
The invention aims to provide a sea rod-shaped bacterium with denitrification and nitrate dissimilarisation reduction into ammonium, a culture method and application thereof, so as to solve the problems of the prior art, realize effective recycling of nitrogen and phosphorus in low-C/N and high-concentration nitrate mariculture tail water, and realize standard discharge of the mariculture tail water in a short time.
In order to achieve the above object, the present invention provides the following solutions:
the present invention provides a strain of bacillus marinus (Marinobacter sp.) which has been deposited in the China general microbiological culture Collection center (ccm) for 10 and 31 of 2022; the preservation address is the microbiological institute of China academy of sciences of national institute of sciences No. 1, 3 of north chen west way, the morning of Beijing city; the preservation number is CGMCC NO.25998.
The invention also provides a culture method of the sea bacillus, which comprises the following steps:
under anaerobic or aerobic conditions, the sea rod-shaped bacteria take nitrate as a nitrogen source to carry out Denitrification (DNF) and nitrate catabolism reduction to ammonium (DNRA) in a heterotrophic culture medium, and a sea rod-shaped bacteria culture product is obtained.
Preferably, in the heterotrophic culture medium, the sea rod-shaped bacteria mainly undergo denitrification under the condition that the C/N is more than or equal to 10, and mainly undergo nitrate catabolism reduction to ammonium under the condition that the C/N is less than or equal to 5.
Preferably, the conditions for denitrification and nitrate catabolism reduction to ammonium are: culturing at 25-35 deg.c and 120-180rpm for 36-72 hr.
The invention also provides a method for constructing an algae-bacteria symbiotic culture system by utilizing the sea bacillus and the sea microalgae, which comprises the following steps:
mixing marine microalgae and sea bacilli according to a volume ratio of 4:1, and culturing to construct a bacteria-algae symbiotic culture system; wherein the marine microalgae is cultivated with an f/2 culture medium; the sea bacillus was cultured with 2216E medium.
Preferably, in the mixed culture system, the biomass ratio of the marine microalgae to the sea bacillus is 1.5-2:1; the biomass ratio is determined by measuring the biomass of microorganisms/microalgae, and then comparing the measured biomass, and the determination of the biomass is a conventional determination method.
The mixed culture conditions are as follows: the illumination intensity is 2000-5000lx at 25-35 ℃, and the stationary culture time is 48h.
Preferably, the marine microalgae comprise any one of Phaeodactylum tricornutum, chlorella seawater, spirulina seawater, chlorella uncinata, etc., and Chlorella occidentalis. The method for recycling nitrogen and phosphorus in the mariculture tail water is a symbiotic method of the sea bacilli and the sea microalgae, the sea bacilli can form symbiotic systems with different classes of sea microalgae, and the sea bacilli can treat NO in the low-C/N mariculture tail water 3 - Rapid conversion of N to NH 4 + N is directly absorbed and utilized by symbiotic microalgae, and the microalgae grow and absorb and utilize phosphate in water.
The invention also provides application of the sea bacillus or the algae symbiotic culture system constructed by the method in sea water culture tail water treatment or effective recycling of nitrogen and phosphorus in sea water culture tail water.
Preferably, the application method comprises the following steps: inoculating the microalgae in the mariculture tail water according to the volume ratio of the algae symbiotic culture system to the mariculture tail water of 1:20, culturing for 4-6 days, and collecting the microalgae in the water body.
The invention discloses the following technical effects:
the invention separates and obtains a strain of sea rod-shaped bacteria which can simultaneously have denitrification and nitrate dissimilation reduction into ammonium, and experiments prove that the sea rod-shaped bacteria can carry out DNRA under the condition of low C/N and preferentially carry out DNF under the condition of high C/N, and the sea rod-shaped bacteria and microalgae are used for symbiotic, so that the effective resource utilization of nitrogen and phosphorus in the sea water of the low C/N and high-concentration nitrate sea water can be realized, and the standard emission of the sea water can be realized in a short time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing characteristics of culture of a culture medium of sea rod-shaped bacteria 2216E;
FIG. 2 is a photograph of a sea rod-shaped bacterium of the present invention by scanning electron microscopy;
FIG. 3 shows the treatment of sea water NO with sea water bacilli under aerobic conditions of different carbon-nitrogen ratios 3 - -N varies with time;
FIG. 4 shows NH of sea water treated by sea bacillus under aerobic condition with different carbon nitrogen ratios 4 + -N varies with time;
FIG. 5 shows the treatment of sea water NO with sea water culture tail water by sea bacillus under anaerobic condition with different carbon nitrogen ratio 3 - -N varies with time;
FIG. 6 shows NH of sea water treated by Haibacterium under anaerobic conditions of different carbon-nitrogen ratios 4 + -N varies with time;
FIG. 7 shows the effect of the symbiotic system of the sea bacillus and the sea chlorella on the recycling of the tail water of the sea water culture;
FIG. 8 shows the effect of the symbiotic system of the sea baculiform and the sea spirulina on the recycling of the sea water culture;
FIG. 9 shows the effect of the symbiotic system of the dinoflagellates such as the sea bacilli and the globes on the recycling of the tail water of mariculture.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The technology for treating the symbiotic wastewater based on the prior algae bacteria has the advantages that: oxygen released by the microalgae through photosynthesis is used by aerobic bacteria/nitrifying bacteria, so that ammonia nitrogen and nitrite nitrogen in the water body are converted, and organic matters in the bacteria decomposed wastewater provide no matters required by the growth of the microalgaeOrganic salts, vitamins, etc., and microalgae can absorb nitrogen and phosphorus nutrient salts through assimilation. The water treatment effect of the algae-bacteria symbiotic system is better than that of an algae-bacteria single system, however, the conventional cognitive symbiotic mode is not suitable for tail water treatment with higher nitrate content in a seawater industrial circulating water culture mode, the symbiotic mode of microalgae and anaerobic denitrifying bacteria is adopted, the denitrification efficiency of denitrifying bacteria can be greatly inhibited by oxygen released by microalgae photosynthesis, and the effective recycling of nitrogen in the tail water can not be realized; the symbiotic mode of microalgae and aerobic denitrifying bacteria has low bacterial denitrification efficiency and cannot realize the recycling of nitrogen. Due to plant properties of microalgae, the microalgae can preferentially utilize NH in water 4 + N is used for self growth, so that microalgae and nitrate are reduced into ammonium (DNRA) bacteria for effectively recycling nitrogen and phosphorus in seawater industrial circulating water culture tail water, and DNRA bacteria are used for recycling NO with high concentration in the tail water 3 - Conversion of N to NH 4 + N is directly absorbed and utilized by microalgae, however, the method has the difficulty that DNRA and Denitrification (DNF) are in two competitive nitrate reduction paths, DNRA generally takes advantage of under the condition of high C/N, DNF processes take advantage of under the condition of low C/N, and DNRA processes are extremely difficult to realize in mariculture tail water with low C/N. Aiming at the defects of the prior art, the invention separates a strain of sea rod-shaped bacteria which can simultaneously have denitrification and nitrate dissimilation reduction into ammonium, and experiments prove that the sea rod-shaped bacteria can carry out DNRA under the condition of low C/N and preferentially carry out DNF under the condition of high C/N, and the sea rod-shaped bacteria and microalgae are used for symbiotic, so that the effective recycling of nitrogen and phosphorus in the sea water of the low C/N and high-concentration nitrate sea water culture can be realized, and the standard emission of the sea water culture tail water can be realized in a short time.
In order to further illustrate the technical effects of the present invention, the following description will be given by way of specific examples:
EXAMPLE 1 isolation and identification of a strain of sea baculiform
The marine corynebacteria (Marinobacter sp.) according to the present invention are isolated from a marine spirulina culture system. The specific separation and identification method comprises the following steps:
(1) The seawater spirulina culture system is scratched and sampled in 2216E solid culture medium, and cultured for 2d at 35 ℃ to obtain the culturable marine bacteria, single bacterial colony is selected to continue to be cultured and purified for multiple times in 2216E solid culture medium to obtain the marine bacteria of single strain, and then the single bacterial colony is selected to be cultured in 2216E liquid culture medium to obtain the purified single bacterial culture solution.
Weighing 52.4g of 2216E solid culture medium powder, heating and dissolving in 1000mL of distilled water, packaging, and sterilizing at 121 ℃ under high pressure for 15 minutes for later use; wherein, 2216E solid culture medium comprises the following components: peptone 5.0 g.L -1 Yeast extract powder 1.0g.L -1 ，FeC 6 H 5 O 7 0.1g·L -1 ，NaCl 19.45g·L -1 ，MgCl 2 5.98g·L -1 ，Na 2 SO 4 3.24g·L -1 ，CaCl 2 1.8g·L -1 ，KCl 2 0.55g·L -1 ，Na 2 CO 3 0.16g·L -1 ，KBr 0.08g·L -1 ，SrCl 2 0.034g·L -1 ，H 3 BO 3 0.022g·L -1 ，Na 2 SiO 3 0.004g·L -1 ，NaF 0.0024g·L -1 ，NaNO 3 0.0016g·L -1 ，Na 2 HPO 4 0.008g·L -1 Agar 15.0 g.L -1 . Culture conditions: the pH is 7.6+/-0.2 at 25 ℃.
The purified bacteria grow on 2216E liquid culture medium, 37.4g of 2216E liquid culture medium powder is weighed, heated and dissolved in 1000mL of distilled water, and autoclaved for 15 minutes at 121 ℃ for standby; wherein, 2216E liquid medium comprises the following components: peptone 5.0 g.L -1 Yeast extract powder 1.0g.L -1 ，FeC 6 H 5 O 7 0.1g·L -1 ，NaCl 19.45g·L -1 ，MgCl 2 5.98g·L -1 ，Na 2 SO 4 3.24g·L -1 ，CaCl 2 1.8g·L -1 ，NaCl 0.55g·L -1 ，Na 2 CO 3 0.16g·L -1 ，KBr0.08g·L -1 ，SrCl 2 0.034g·L -1 ，H 3 BO 3 0.022g·L -1 ，Na 2 SiO 3 0.004g·L -1 ，NaF 0.0024g·L -1 ，NaNO 3 0.0016g·L -1 ，Na 2 HPO 4 0.008g·L -1 . Culture conditions: the pH is 7.6+/-0.2 at 25 ℃.
(2) Culture characteristics
Sea bacillus (Marinobacter sp.) gram-negative bacteria, facultative aerobiotic, rod-shaped cells, and single flagellum, which can swim. Culturing on 2216E solid culture medium at 35deg.C for 2d, and the colony is yellowish, semitransparent, slightly convex in the middle, rough in surface, regular round, irregular in edge, and about 1.0-2.0mm in diameter. The pH required by the growth of the strain is 7.0-9.0, and the optimal pH is 8.0.
(3) Results of Gene identification
The 16S rRNA gene sequence was determined. PCR amplification, sequencing and BLAST alignment of the 16S rRNA gene sequence of Bacillus marinus (Marinobacter sp.) revealed that the similarity of the 16S rRNA gene sequence of Bacillus marinus (Marinobacter sp.) to Marinobacter sp.SL013A-34A-1, marinobacter sp.Sw0106-07 and Marinobacter hydrocarbonoclasticus strain RMR was 99.93%.
The invention is identified to obtain a strain of sea bacillus (Marinobacter sp.) which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in 10 months and 31 days of 2022; the preservation address is the microbiological institute of China academy of sciences of national institute of sciences No. 1, 3 of north chen west way, the morning of Beijing city; the preservation number is CGMCC NO.25998.
EXAMPLE 2 functional verification of sea bacilli
1. Verifying that the sea bacillus has the functions of denitrification and nitrate dissimilation reduction to ammonium at the same time
1.1 aerobic culture
Inoculating the strain of the sea bacillus into a heterotrophic culture medium under aerobic conditions (DO >6 mg/L), culturing the strain of the sea bacillus under the conditions that the pH is 6-9 and the rotation speed of a constant-temperature shaking table is 120-180rpm at 25-35 ℃ (any conditions within the range can realize the same technical effect); wherein, the heterotrophic culture medium adopts the following two culture mediums with different C/N values for experiments:
seawater culture tail water reverseThe components of the nitrifying culture medium are as follows: naNO 3 0.607g/L，KH 2 PO 4 0.044g/L，C 6 H 12 O 6 2.500g/L, 1L of mariculture tail water; the culture medium is sterilized before use.
The nitrate in the tail water of mariculture is reduced to an ammonium culture medium component by differentiation: naNO 3 0.607g/L，KH 2 PO 4 0.044g/L，C 6 H 12 O 6 1.250g/L, and the culture medium is sterilized before use.
As shown in fig. 3 and 4, the results show that: under the aerobic condition of C/N=10, more than 80% of NO in the culture tail water can be realized after 36 hours of treatment 3 - -removal of N; 72h NO 3 - N removal rate 94.65% (FIG. 3), NH 4 + N is not accumulated (fig. 4); under the aerobic condition of C/N=5, NO is treated for 36 hours due to insufficient carbon source 3 - -a reduction in N of about 50mg/L;72h NO 3 - The N removal was only 58.4% (FIG. 3), but about 42.85mg/L NH occurred 4 + N accumulation (fig. 4).
1.2 anaerobic culture
Under anaerobic condition (dissolved oxygen content DO <2 mg/L), the strain of sea bacillus is inoculated into heterotrophic culture medium, pH is 6-9, and culture is carried out under the condition of rotating speed of constant-temperature shaking table 120-180rpm at 25-35 ℃ (the same technical effect can be realized under any condition within the range). Wherein, the heterotrophic culture medium adopts the following two culture mediums with different C/N values for experiments:
the seawater culture tail water denitrification culture medium comprises the following components: naNO 3 0.607g/L，KH 2 PO 4 0.044g/L，C 6 H 12 O 6 2.500g/L, 1L of mariculture tail water; the culture medium is sterilized before use. Wherein, the sea culture tail water is prepared by adding the following concentration components into sea water: naNO 3 0.607g/L，KH 2 PO 4 0.044g/L，C 6 H 12 O 6 2.500g/L; to simulate sea water aquaculture tail water.
The nitrate in the tail water of mariculture is reduced to an ammonium culture medium component by differentiation: naNO 3 0.607g/L，KH 2 PO 4 0.044g/L，C 6 H 12 O 6 1.250g/L, 1L of mariculture tail water; the culture medium is sterilized before use.
As shown in fig. 5 and 6, the results show that: under the anaerobic condition of C/N=10, the NO in the culture tail water can be more than 80% after 48 hours of treatment 3 - Removal of N, no NH occurs 4 + -accumulation of N; 72h NO 3 - N removal of 90.70% (FIG. 5), likewise NH no longer occurs 4 + -accumulation of N; under anaerobic condition with C/n=5, after 48h of treatment, NO 3 - -a reduction in N of about 50mg/L;72h NO 3 - N removal was only 49.18% (FIG. 5), but about 32.15mg/L NH occurred 4 + N accumulation (fig. 6).
Under heterotrophic conditions, the bacillus marinus strain can be used for removing nitrate and organic matters in mariculture tail water; in the denitrification process of the high-concentration nitrate culture tail water, the sea bacillus uses organic carbon as an electron donor to reduce the high-concentration nitrate in the culture tail water, so that NO in the culture tail water can be rapidly reduced 3 - -N content. The above results illustrate: the sea bacillus can realize NO in the sea water culture tail water under both aerobic and anaerobic conditions 3 - Removal of N, but NH can be achieved only at low C/N conditions 4 + Accumulation of N.
Example 3 utilization of a Bacillus marinus Strain having both denitrification and nitrate differentiation reduction to ammonium in seawater Tail Water Nitrogen and phosphorus Recycling
1. Method for constructing algae-bacteria symbiotic culture system by using sea bacillus and sea microalgae
Taking chlorella (green alga), spirulina (blue alga), and dinoflagellates (golden alga) as examples, constructing an alga-bacterium symbiotic culture system with marine microalgae.
50mL of sea bacillus culture solution OD is added into 200mL of sea microalgae culture system 750 0.600-0.800 (preferably-0.800), culture solution OD of sea bacillus 600 0.800-1.000 (preferably 1.000) in microalgae culture systemPerforming mixed culture, namely, the biomass ratio of microalgae to bacteria is 1.5-2:1 (namely, comparison is performed after the biomass of the microalgae and the microorganisms is measured, and the preferable ratio is 1.5:1); the culture condition is 25-35 ℃ (preferably 35 ℃), the illumination intensity is 2000-5000lx (preferably 4000 lx), the shaking is not needed, the culture time is 48 hours, and the construction of the algae-bacteria symbiotic system is completed.
The method for obtaining the marine microalgae culture system comprises the following steps: the marine microalgae grows in an f/2 culture medium, wherein the f/2 culture medium comprises the following components: naNO 3 75.0mg·L -1 ，NaH 2 PO 4 ·H 2 O 5.0mg·L -1 Trace elements (CuSO) 4 ·5H 2 O 0.0098mg·L -1 ，ZnSO 4 ·7H 2 O 0.022mg·L -1 ，CoCl 2 ·6H 2 O 0.01mg·L -1 ，MnCl 2 ·4H 2 O 0.18mg·L -1 ，Na 2 MoO 4 ·2H 2 O 0.0063mg·L -1 ，Na 2 EDTA0.00436mg·L -1 ，FeCl 3 ·6H 2 O 0.00315mg·L -1 )，Vitamin(B 1 0.1mg·L -1 ，Biotin 0.0005mg·L -1 ，B 12 0.0005mg·L -1 ). The culture condition is that the illumination culture is carried out at 30 ℃, the illumination intensity is 4000lx, and the light-dark period is 12h to 12h.
The method for obtaining the culture solution of the sea bacillus comprises the following steps: purified bacteria grow on 2216E liquid culture medium, 37.4g of 2216E liquid culture medium powder is weighed, heated and dissolved in 1000mL of distilled water, and the mixture is sterilized at 121 ℃ for 15 minutes for standby; wherein, 2216E liquid medium comprises the following components: peptone 5.0 g.L -1 Yeast extract powder 1.0g.L -1 ，FeC 6 H 5 O 7 0.1g·L -1 ，NaCl 19.45g·L -1 ，MgCl 2 5.98g·L -1 ，Na 2 SO 4 3.24g·L -1 ，CaCl 2 1.8g·L -1 ，NaCl 0.55g·L -1 ，Na 2 CO 3 0.16g·L -1 ，KBr 0.08g·L -1 ，SrCl 2 0.034g·L -1 ，H 3 BO 3 0.022g·L -1 ，Na 2 SiO 3 0.004g·L -1 ，NaF 0.0024g·L -1 ，NaNO 3 0.0016g·L -1 ，Na 2 HPO 4 0.008g·L -1 . At 25℃the pH was 7.6.+ -. 0.2. The culture conditions were 35℃and the shaking speed was 150rpm.
2. The algae-bacteria symbiotic culture system is applied to the treatment of the seawater culture tail water, and the specific method is as follows:
inoculating in the mariculture tail water according to the volume ratio of the algae-bacteria symbiotic culture system to the mariculture tail water of 1:200; culturing for 4-6 days (preferably 5 days) after 25-35 ℃ (preferably 25 ℃) illumination 2000lx,12h illumination/12 h darkness, and harvesting microalgae in the water body, so that the treatment of the seawater culture tail water can be completed, and meanwhile, the effective recycling of nitrogen and phosphorus in the seawater culture tail water is realized;
wherein, the load of mariculture tail water is: NO (NO) 3 - -N 50mg/L，PO 4 3- P20 mg/L, dissolved organic carbon DOC30mg/L.
As shown in figures 7-9, after symbiotic with marine microalgae, the symbiotic system can be used for recycling nitrogen and phosphorus in the seawater culture tail water, and after 96 hours, the symbiotic system can realize the total absorption of nitrogen and phosphorus in the seawater culture tail water, wherein the fastest speed is that of a seawater chlorella system, and the ratio of 100mg/L NO 3 - the-N can be completely removed after 72h, 20mg/L PO 4 3- P can be fully absorbed and utilized only by 36h (fig. 7); secondly, a seawater spirulina system is adopted, and the concentration of NO is 100mg/L 3 - the-N can be completely removed after 84 hours, 20mg/L PO 4 3- P can reach full absorption utilization at 48h (fig. 8); 100mg/L NO in dinoflagellate system 3 - Complete removal of 20mg/L PO can be achieved at 96h for-N 4 3- Complete removal of P will be achieved at 60h (fig. 9).
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. Sea bacillusMarinobacter sp.) The method is characterized in that the preservation number of the sea bacillus is CGMCC No.25998.
2. The method for culturing a sea rod bacterium according to claim 1, comprising: under the anaerobic or aerobic condition, the sea rod-shaped bacteria take nitrate as a nitrogen source to carry out denitrification and nitrate catabolism reduction to ammonium in a heterotrophic culture medium, and a sea rod-shaped bacteria culture product is obtained.
3. The method according to claim 2, wherein the culture medium is one in which the sea rod is mainly denitrified under the condition that C/N is not less than 10 and the sea rod is reduced to ammonium under the condition that C/N is not more than 5.
4. The method of claim 2, wherein the conditions for denitrification and nitrate catabolism reduction to ammonium are: culturing at 25-35 deg.c and 120-180rpm for 36-72 hr.
5. The method for constructing an algae-bacteria symbiotic culture system by utilizing the sea bacillus and the sea microalgae according to claim 1, which is characterized by comprising the following steps: mixing marine microalgae and sea bacilli according to a volume ratio of 4:1, and culturing to construct a bacteria-algae symbiotic culture system; wherein the marine microalgae is cultivated with an f/2 culture medium; culturing the sea bacillus with 2216E culture medium;
the marine microalgae is Chlorella vulgaris, spirulina or Chlorella vulgaris.
6. The method of claim 5, wherein the biomass ratio of marine microalgae to sea bacilli in the mixed culture system is 1.5-2:1;
the mixed culture conditions are as follows: the illumination intensity is 2000-5000lx at 25-35 ℃, and the stationary culture time is 48h.
7. Use of a corynebacterium marinum according to claim 1 or a symbiotic culture system of algae constructed by the method according to any one of claims 5-6 in the treatment of mariculture tail water or the effective recycling of nitrogen and phosphorus in mariculture tail water.
8. The application according to claim 7, wherein the application method is: inoculating the microalgae in the mariculture tail water according to the volume ratio of the algae symbiotic culture system to the mariculture tail water of 1:20, culturing for 4-6 days, and collecting the microalgae in the water body.
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