CN113913308B - Marine rhodotorula strain WCBC capable of efficiently removing ammonium salt and application thereof - Google Patents

Marine rhodotorula strain WCBC capable of efficiently removing ammonium salt and application thereof Download PDF

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CN113913308B
CN113913308B CN202110911129.8A CN202110911129A CN113913308B CN 113913308 B CN113913308 B CN 113913308B CN 202110911129 A CN202110911129 A CN 202110911129A CN 113913308 B CN113913308 B CN 113913308B
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rhodotorula
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ammonium salt
nitrite
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徐炜
左啸天
张新旭
高渊皓
骆祝华
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Shenzhen University
Third Institute of Oceanography MNR
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F2101/166—Nitrites
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    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract

The invention relates to the field of microorganisms, in particular to a marine rhodotorula strain WCBC capable of efficiently removing ammonium salt and application thereof. The invention provides a rhodotorula (Rhodotorula diobovata) WCBC, the preservation number of which is CCTCC NO: m2021966. The rhodotorula (Rhodotorula diobovata) WCBC provided by the invention can utilize a plurality of different inorganic nitrogen sources to grow and remove nitrogen, can effectively remove nitrate, nitrite and ammonium salt in the trinitrogen wastewater, and reduces the total nitrogen content of the wastewater in a mode of partially converting the nitrogen sources into gaseous nitrogen.

Description

Marine rhodotorula strain WCBC capable of efficiently removing ammonium salt and application thereof
Technical Field
The invention relates to the field of microorganisms, in particular to a marine rhodotorula strain WCBC capable of efficiently removing ammonium salt and application thereof.
Background
In recent years, with the development of industry and agriculture, industrial production activities using inorganic nitrogen as a raw material or an auxiliary material, and excessive application of nitrogen-containing fertilizers in soil, have resulted in more and more "tri-nitrogen" pollution.
The "triazo" pollution is a collective term for nitrate, nitrite and ammonium salt pollution. The 'tri-nitrogen' pollution brings a plurality of damages to natural environment and human society, and causes water eutrophication when serious, red tide, water bloom and other phenomena are generated, the ecological balance of the water body is seriously destroyed, and huge economic loss is caused for related industries. In addition, "triazene" contamination is also a great hazard to humans. Nitrite in the triazene can induce methemoglobin blood disease, cancer and the like when serious after being ingested into human body, and has great influence on human health. Ammonium salt and nitrate in the triazo can be partially converted into nitrite through in-vivo nitrosation after being finally taken into a human body, and the triazo is also not beneficial to the health of the human body.
At present, the related national standard prescribes the concentration of the tri-nitrogen in the water body, wherein the emission standard GB/T16171-2012 of the coking chemical industry prescribes that the direct emission limit value (calculated by N) of the water pollutants of ammonia nitrogen and total nitrogen in the existing enterprises is 15mg/L, 30mg/L and the indirect emission limit value is 25mg/L and 50mg/L. Sanitary standard GB/T5749-2006 specifies that the highest limit values (calculated by N) of water quality indexes of nitrate, nitrite and ammonium salt of drinking water are 10mg/L, 1mg/L and 0.5mg/L. The quality standard GB/T14848-2017 of underground water prescribes that III water quality suitable for centralized living drinking water digging and industrial and agricultural water should meet the requirements of nitrate, nitrite and ammonium salt of less than 20mg/L, 1mg/L and 0.5mg/L. Therefore, the method effectively treats the 'tri-nitrogen' pollution and is one of the key points in the water pollution treatment.
In recent years, three types of treatment methods, namely physical methods, chemical methods and biological methods, are mainly proposed for 'triazo' pollution. Physical and chemical methods tend to have the disadvantages of higher investment costs and less stable effects. In contrast, biological methods are the most widely used, and various microorganisms which exist in nature and can utilize inorganic nitrogen are used for treating nitrogen-containing wastewater, so that the method has the advantages of low cost, mild conditions and the like. However, most microorganisms often only use one or two inorganic nitrogen, and do not have the capability of using nitrate, nitrite and ammonium salt at the same time, so that sewage with the three nitrogen not reaching standards is treated by using a biological method, and nitrogen removal is often needed by using a plurality of strains under different conditions. The method is used for searching the bacterial strain which can use nitrate, nitrite and ammonium salt as nitrogen sources at the same time and utilizing the bacterial strain, and is one solution for treating the tri-nitrogen pollution of the water body by a biological method.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defect of narrow range of available inorganic nitrogen sources of microorganisms in the prior art, thereby providing a marine rhodotorula strain WCBC for efficiently removing ammonium salt and application thereof.
Rhodotorula (Rhodotorula diobovata) WCBC with a preservation number of CCTCC NO: m2021966.
A microbial inoculum contains the above metabolites of Rhodotorula WCBC and/or Rhodotorula WCBC as active ingredients.
The application of the rhodotorula WCBC or the microbial inoculum in nitrogen removal of the waste water containing the three nitrogen is provided.
Optionally, the tri-nitrogen is ammonium nitrogen, nitrate nitrogen or nitrite nitrogen.
The application of the rhodotorula WCBC or the microbial inoculum in nitrogen conversion.
Optionally, the nitrogen conversion is specifically nitrification and/or nitrogen assimilation.
Rhodotorula WCBC was deposited in China center for type culture Collection, accession number: the mail code 430072 of the university of Wuhan in China has the preservation number of: cctccc NO: m2021966, classified as Rhodotorula diobovata WCBC, the detection result of this strain was survival.
Rhodotorula (Rhodotorula diobovata) WCBC, referred to herein as Rhodotorula WCBC.
The technical scheme of the invention has the following advantages:
1. the rhodotorula (Rhodotorula diobovata) WCBC provided by the invention can utilize a plurality of different inorganic nitrogen sources to grow and remove nitrogen, can effectively remove nitrate, nitrite and ammonium salt in the trinitrogen wastewater, and reduces the total nitrogen content of the wastewater in a mode of partially converting the nitrogen sources into gaseous nitrogen.
2. According to the application of the rhodotorula (Rhodotorula diobovata) WCBC provided by the invention, nitrogen can be removed from nitrogen-containing wastewater polluted by different inorganic nitrogen by removing nitrogen from the rhodotorula (Rhodotorula diobovata) WCBC provided by the invention in the nitrogen removal of the waste water, so that the development of treatment of the nitrogen pollution is promoted.
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 description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a colony of the WCBC of rhodotorula in example 1, the left view is the front side, the right view is the back side;
FIG. 2 is a phylogenetic tree of the strain of example 1; strain WCBC represents Rhodotorula WCBC;
FIG. 3 shows the use of KNO in example 2 3 Or NaNO 2 Or (NH) 4 ) 2 SO 4 A growth curve as the sole nitrogen source;
FIG. 4 is the test results of the nitrite free property in example 2;
FIG. 5 is the test results of the nitrite removal characteristic of example 2;
FIG. 6 shows the results of the test for the deamination characteristics of example 2.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any person who may, in the light of the present invention or in combination with other prior art features, come within the scope of the invention.
The specific experimental procedures or conditions are not noted in the examples and may be performed according to the procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
The liquid medium and the solid medium used in example 1 below satisfy the following conditions:
the liquid medium per liter comprises: naNO 3 0.3641g or NaNO 2 0.2956g or (NH) 4 ) 2 SO 4 0.5661g、CH 3 COONa·3H 2 O 7.85g、Na 2 HPO 4 ·12H 2 O 6.67g、KH 2 PO 4 1.5g、 MgSO 4 ·7H 2 O0.1 g and 2ml of microelement solution.
Wherein, per liter of solid medium comprises: naNO 3 0.3641g or NaNO 2 0.2956g or (NH) 4 ) 2 SO 4 0.5661g、CH 3 COONa·3H 2 O 7.85g、Na 2 HPO 4 ·12H 2 O 6.67g、KH 2 PO 4 1.5g、MgSO 4 ·7H 2 0.1g of O, 2ml of trace element solution and 18g of agar.
Each liter of microelement solution comprises Na 2 EDTA 63.70g,CaCl 2 5.50g,ZnSO 4 ·7H 2 O 3.90g, MnCl 2 ·4H 2 O 5.06g，FeSO 4 ·7H 2 O 5.00g,Na 2 MoO 4 ·2H 2 O 1.00g,CuSO 4 1.01g, CoCl 2 ·6H 2 O1.61 g, vitamin B12 0.01mg.
EXAMPLE 1 isolation and identification of Rhodotorula WCBC
This example relates to a nitrifying and nitrogen assimilating fungus, whose isolation and identification steps are as follows:
1. isolation of strains
Transferring 1mL ocean surface seawater into a liquid culture medium (formula is shown above) taking nitrate, nitrite or ammonium salt as a unique nitrogen source for enrichment for 2-3 weeks, diluting 1mL of enrichment culture medium 100 times, coating and inoculating onto a solid culture medium (namely the solid culture medium above), culturing for 4-5 days, growing single bacterial colony, selecting single bacterial colony, transferring the single bacterial colony into a new solid culture medium for streak culture, selecting streak again after the new single bacterial colony grows out, and repeating the steps for 2-3 times to finally obtain the pure culture of the bacterial strain.
On a solid culture medium, the strain can grow in a large quantity after 4-5 days of growth, the whole colony is irregularly round and apricot red (see left diagram of figure 1), and the colony is thick, compact, and wet and sticky on the surface. The colony centers were micro-elevated. The back (see right figure of figure 1) is brown yellow, and the surface is flat and smooth without wrinkles.
2. Identification of strains
The pure culture of the strain was scraped off,taking the thallus and utilizing FastDNA TM SPIN Kit for Soil the kit extracts the DNA of the strain. The extracted DNA was amplified on a thermal cycler for the ITS sequence of the genome by primers of ITS4 (SEQ ID NO:2: 5'-TCCGTAGGTGAACCTGCGG-3') and ITS5 (SEQ ID NO:3: 5'-TCCTCCGCTTATTGATATGC-3'), the length of the sequence being about 600bp. The PCR product of the amplified ITS sequence (SEQ ID NO: 1) entrusts Xiaomen platinum Rayleigh sequencing company to carry out 26SrDNA-ITS region gene sequence analysis, after the sequencing is successful, the redundant sequences at the front end and the rear end of the ITS sequence of the fungi are removed by using BioEdit software, the sequences with tidy peaks are reserved, then Blast analysis and comparison are carried out on the ITS sequence of the fungi on NCBI website (www.ncbi.nlm.nih.gov /), and the species position is determined according to the sequence similarity of 99-100% as a standard.
The comparison result shows that: the ITS sequence (SEQ ID NO: 1) of the strain has the highest similarity with Rhodotorula diobovata CBS 6085 (accession number NR _ 073271.1) of 99.64%, and the strain is determined to belong to Rhodotorula diobovata in taxonomy according to the phylogenetic tree result and morphological characteristics and is fungus. The phylogenetic tree of the strain is shown in figure 2, and the strain 2021 is preserved in China center for type culture Collection, with a preservation address: the university of Wuhan in China, post code 430072, storage number is: cctccc NO: m2021966, class name Rhodotorula diobovata WCBC.
Example 2 detection of the ability of Rhodotorula WCBC to remove nitrate, nitrite and ammonium salts from wastewater
1.1 Pre-culture
Rhodotorula WCBC isolated in example 1 was shake-cultured for 5 days using 100mL of liquid medium at 28℃and a rotational speed of 120rpm.
1.2 growth experiments with different Nitrogen sources
The denitrification liquid medium comprises per liter: KNO (KNO) 3 0.58g or NaNO 2 0.3958g or (NH) 4 ) 2 SO 4 0.3790g, glucose 1.6g, na 2 HPO 4 ·12H 2 O 6.67g、KH 2 PO 4 1.5g、MgSO 4 ·7H 2 O0.1 and g, naCl 30g and 2ml of microelement solution with pH of 6.5. Per liter microThe volume element solution comprises Na 2 EDTA 63.70g,CaCl 2 5.50g,ZnSO 4 ·7H 2 O 3.90g,MnCl 2 ·4H 2 O 5.06g，FeSO 4 ·7H 2 O 5.00 g,Na 2 MoO 4 ·2H 2 O 1.00g,CuSO 4 1.01g,CoCl 2 ·6H 2 O1.61 g, vitamin B12 0.01mg.
In the growth experiments of different nitrogen sources, KNO is respectively used 3 Or NaNO 2 Or (NH) 4 ) 2 SO 4 As a sole nitrogen source, the denitrification liquid culture medium is sterilized at 115 ℃ for 20min and then inoculated with 1mL of OD 600 The red yeast WCBC preculture adjusted to 0.4 is placed in a shaking table at 28 ℃ and 120rpm for culturing for 120 hours. Detecting OD reflecting strain growth condition in culture medium after culture 600 Values.
The detection results are shown in FIG. 3.
1.3 test for nitrite-removing Properties
Selecting nitrate (KNO) 3 ) The denitrification experiment was performed according to the denitrification liquid medium and the culture conditions described in 1.2 as the only nitrogen source. Detecting nitrate, nitrite, ammonium and OD in the culture medium every 12h during culture 600 。
In the experiment, the detection of nitrate, nitrite and ammonium salt is respectively carried out according to GB/T12763.4-2007 sea investigation Specification 4: the zinc-cadmium reduction method, the diazo azo method and the sodium hypobromite oxidation method in the seawater chemical element survey. Efficiency of nitrite removal = (content of nitrate in initial Medium-content of nitrate in Medium at termination)/content of nitrate in initial Medium × 100%
The detection results are shown in FIG. 4.
The results show that: in the process of the nitrite removal of the rhodotorula WCBC, after 48 hours of culture, the rhodotorula WCBC starts to enter a logarithmic growth phase, starts to obviously consume nitrate in a culture medium, and after 120 hours of culture is finished, the nitrite removal efficiency is 51.15%, which indicates that the rhodotorula WCBC can effectively perform the nitrite removal. During the incubation period, there was no significant nitrite accumulation, and occasionally slight ammonium accumulation.
1.4 test for nitrite-removing Properties
Selecting nitrite (NaNO) 2 ) The denitrification experiment was performed according to the denitrification liquid medium and cultivation conditions described in 1.2 as the sole nitrogen source. The nitrate, nitrite, ammonium and OD in the culture medium were detected every 12h during the culture according to 1.3 600 . Efficiency of nitrite removal = (nitrite content in initial Medium-nitrite content in Medium at termination)/nitrite content in initial Medium × 100%
The detection results are shown in FIG. 5.
The results show that: in the process of removing nitrite, after the rhodotorula WCBC is cultured for 72 hours, the rhodotorula WCBC starts to enter a logarithmic growth phase, and after the culture is finished for 120 hours, the nitrite removing efficiency is 32.34%, which indicates that the rhodotorula WCBC can effectively remove nitrite. During the culture period, no obvious nitrate accumulation and ammonium salt accumulation exist.
1.5 De-ammonium salt Property experiment
Selecting ammonium salts ((NH) 4 ) 2 SO 4 ) The denitrification experiment was performed according to the denitrification liquid medium and cultivation conditions described in 1.2 as the sole nitrogen source. The nitrate, nitrite, ammonium and OD in the culture medium were detected every 12h during the culture according to 1.3 600 . Efficiency of deamination = (content of ammonium salt in initial Medium-content of ammonium salt in Medium at termination)/content of ammonium salt in Medium at initial × 100%
The detection results are shown in FIG. 6.
The results show that: in the process of removing ammonium salt from the rhodotorula WCBC, after 48 hours of culture, the rhodotorula WCBC starts to enter a logarithmic growth phase, and starts to obviously consume ammonium salt in a culture medium, and after 120 hours of culture is finished, the ammonium salt removing efficiency is 86.25%, which indicates that the rhodotorula WCBC can effectively remove ammonium salt. During the culture period, obvious nitrate accumulation exists, which indicates that the rhodotorula WCBC has the nitrification capability.
1.6 total Nitrogen removal Property experiment
Selecting ammonium salt (((NH) 4 ) 2 SO 4 ) Nitrite (NaNO) 2 ) Respectively the only nitrogen source is used for the preparation of the nitrogen source,preparing a denitrification liquid culture medium, wherein each liter of denitrification liquid culture medium comprises: naNO 2 0.3450g or (NH) 4 ) 2 SO 4 0.3300g, glucose 1.6g, na 2 HPO 4 ·12H 2 O 6.67g、KH 2 PO 4 1.5g、MgSO 4 ·7H 2 O0.1 g, naCl 30g and 2ml of microelement solution, the pH is 6.5. Each liter of microelement solution comprises Na 2 EDTA 63.70g, CaCl 2 5.50g,ZnSO 4 ·7H 2 O 3.90g,MnCl 2 ·4H 2 O 5.06g，FeSO 4 ·7H 2 O 5.00g, Na 2 MoO 4 ·2H 2 O 1.00g,CuSO 4 1.01g,CoCl 2 ·6H 2 O1.61 g, vitamin B12 0.01mg.
Sterilizing the denitrification liquid culture medium at 115 deg.C for 20min, inoculating 1mL OD 600 The red yeast WCBC preculture adjusted to 0.4 is placed in a shaking table at 28 ℃ and 120rpm for culturing for 120 hours. Total nitrogen removal characterization experiments were performed and nitrogen balance analysis was performed. Specifically, total nitrogen in the medium, initial intracellular nitrogen, total nitrogen in the medium at the time of termination, intracellular nitrogen at the time of termination, gaseous nitrogen were detected for 0h during the culture.
The test results are shown in Table 1.
TABLE 1 total nitrogen removal experimental results under different Nitrogen Source conditions
Figure SMS_1
Note that: the initial total nitrogen/total nitrogen in the initial medium in the above table is the actual measurement.
The results show that: in the process of total nitrogen removal, the total nitrogen in the culture medium is reduced by 33.3mg/L when the ammonium salt is taken as the sole nitrogen source, wherein 82.43 percent of the total nitrogen is converted into bacterial strain biomass, and 17.57 percent of the total nitrogen is converted into gaseous nitrogen which is discharged from the culture system to the atmosphere. With nitrite as the sole nitrogen source, the total nitrogen in the medium was reduced by 16.67mg/L, 92.86% of which was converted to strain biomass and 7.79% of which was converted to gaseous nitrogen which was vented from the culture system to atmosphere. Experiments show that the rhodotorula WCBC mainly has two types of nitrogen removal related paths, wherein nitrogen assimilation is dominant in the nitrogen removal process, and nitrification is the second.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While obvious variations or modifications of the invention are contemplated as falling within the scope of the present invention.
Sequence listing
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Claims (6)

1. Rhodotorula (Rhodotorula diobovata) WCBC with a preservation number of CCTCC NO: m2021966.
2. A microbial inoculum, wherein the active ingredient is rhodotorula (Rhodotorula diobovata) WCBC according to claim 1.
3. Use of the rhodotorula (Rhodotorula diobovata) WCBC according to claim 1 or the microbial inoculum according to claim 2 for nitrogen removal of waste water from triazenes.
4. Use according to claim 3, characterized in that the tri-nitrogen is ammonium nitrogen, nitrate nitrogen or nitrite nitrogen.
5. Use of a rhodotorula (Rhodotorula diobovata) WCBC according to claim 1 or a microbial inoculum according to claim 2 for nitrogen conversion.
6. Use according to claim 5, characterized in that the nitrogen conversion is in particular nitrification and/or nitrogen assimilation.
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