CN111996131B

CN111996131B - Pichia pastoris of shigella delavayi for degrading ammonia nitrogen and application

Info

Publication number: CN111996131B
Application number: CN202010928211.7A
Authority: CN
Inventors: 蒋承建; 于苒; 何升; 阎冰; 莫雪艳; 惠秦妍; 蔡杏华; 卜孺; 孙会杰; 陈思
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2021-06-25
Anticipated expiration: 2040-09-07
Also published as: CN111996131A

Abstract

The invention belongs to the technical field of industrial microorganisms, and relates to pichia pastoris of deprenwei for degrading ammonia nitrogen and application thereof. The aroma-producing Pichia de-rienzi yeast for degrading ammonia nitrogen is named as Pichia de-rienzi yeast HJ2, and is deposited in China general microbiological culture Collection center in 2020, 06-01.01.numbered CGMCC No. 19897. The Pichia kudriavzevii provided by the invention can independently utilize organic matters of pentose and hexose to generate rich aromatic compounds, is salt-resistant and heavy metal-resistant, can adapt to a wide pH range, and expands the environmental conditions for treating wastewater containing ammonia nitrogen. The strain is used for treating the water body containing high-concentration ammonia nitrogen, the production process is environment-friendly, the operation is simple, no secondary pollution is caused, and the strain is very suitable for industrial large-scale production.

Description

Pichia pastoris of shigella delavayi for degrading ammonia nitrogen and application

Technical Field

The invention belongs to the technical field of industrial microorganisms, and relates to pichia pastoris of deprenwei for degrading ammonia nitrogen and application thereof.

Background

The nitrogen element in the ammonia nitrogen wastewater is ammonium ion (NH)₄ ⁺) And free ammonia (NH)₃) Is in the form of a solution of (a) in water,a large amount of ammonia nitrogen wastewater can be generated in domestic sewage and landfill leachate, and industrial production such as steel, oil refining, petrochemical industry, glass manufacturing, chemical fertilizers, tanning, feed production and the like, the sources of the ammonia nitrogen wastewater are very wide, the eutrophication of water bodies can be caused by the high oxygen consumption of ammonia nitrogen in the wastewater, water bloom and red tide are formed to damage the water body environment, the toxic action is generated on aquatic organisms, and the life safety can be endangered when people eat polluted aquatic products by mistake.

In recent years, the problem of water pollution caused by ammonia nitrogen discharge has attracted wide attention at home and abroad. With the aggravation of water resource crisis and the deeper understanding of people on ammonia nitrogen wastewater, the comprehensive treatment of ammonia nitrogen wastewater by searching economic and efficient treatment technology becomes a problem to be solved urgently.

The biological method is used as a sewage treatment process and mainly comprises an anaerobic biological treatment technology, an aerobic biological treatment technology and a natural biological treatment technology because the biological method has the characteristics of small occupied area, strong treatment capacity, small environmental influence factors and the like. Compared with other treatment methods, the microbial degradation of organic matters has incomparable advantages. The micromolecule organic matter generated after the ring opening and chain breaking are subjected to physicochemical treatment is suitable for biological removal, and can reduce the treatment energy consumption and the operation cost. Biological methods have become the main technology for treating livestock and poultry breeding wastewater in recent years.

The aroma-producing yeast is also called ester-producing yeast, is a general name of yeast with strong ester-producing capability, generally refers to a kind of yeast which can generate various aroma components represented by esters or precursor substances thereof in the growth and metabolism process, mainly produces ethyl acetate, belongs to membrane-producing yeast, and comprises candida, hansenula, pichia, torulopsis, and the like. The aroma-producing yeast is a yeast which can be metabolized and synthesized into esters as a main part and can promote the generation of volatile flavor substances such as alcohol, aldehyde, phenol and the like (reference document: screening of salt-tolerant aroma-producing yeast in Pendong high-salt dilute soy sauce mash and aroma-producing characteristic research). In recent years, the method is widely applied to various fields related to food fermentation due to the special physiological properties of aroma production, including flavor-oriented industries such as tobacco flavor, soy and vinegar brewing, traditional wheaten food, fruit wine sauce fermentation, white spirit brewing and the like, plays a role in enriching aroma and improving sensory quality, and has the potential of improving the sensory quality of wine. Currently, no Pichia pastoris strain capable of degrading ammonia nitrogen has been reported.

Disclosure of Invention

In view of the above, the invention aims to provide a bacterial strain for degrading ammonia nitrogen, which can not only increase flavor nutrient substances in food fermentation, but also improve the biological removal efficiency of ammonia nitrogen-containing wastewater, and meet the large-scale industrial application of ammonia nitrogen degradation by a biological method.

The aroma-producing Pichia de-rienzi yeast for degrading ammonia nitrogen is named as Pichia de-rienzi yeast HJ2, and is deposited in China general microbiological culture Collection center in 2020, 06-01.01.numbered CGMCC No. 19897.

Furthermore, the growth temperature range of the strain is 15-48 ℃, the growth pH range is 2.0-9.0, and the weight content of sodium chloride in a growth medium is 0-9%.

Furthermore, the optimal growth temperature of the strain is 37 ℃, and the optimal growth pH is 3.0-8.0.

Further, the fermentation time of the strain is 12-168 hours.

The carbon source assimilable by the strain provided by the invention comprises glucose, sucrose, maltose, D-fructose, D-mannitol, D-galactose, L-arabinose, D-sorbitol, xylose, glycerol, soluble starch and non-assimilable lactose.

The strain provided by the invention can resist heavy metals of Cr, Cu, Mn, Co, Ni and Zn, and has strong tolerance to heavy metal environments with concentrations of 0-24ppm, 0-640ppm, 0-5494ppm, 0-118ppm, 0-295ppm and 0-654ppm respectively.

The variety quantity of the flavor metabolites obtained by fermenting glucose for 12-168 hours by the strain provided by the invention is 40-62%, and the content of flavor substances is 32.46-90.22%.

The aroma metabolites comprise esters, aldehydes, ketones, furans, acids and alcohols, the aroma components comprise isoamyl acetate, 4-methylbenzaldehyde, phenethyl alcohol, isovaleraldehyde, phenethyl acetate, diisobutyl phthalate, octanoic acid, (E) -3-decenoic acid, 2, 6-bis (tert-butyl) phenol, 1, 2-bis (2-methylpropyl) phthalate, farnesol, coumarin, indole, levogyration, beta-dihydro-beta-ionone, linolenic acid, lupinene, nerolidol and alpha-caryophyllene.

The second purpose of the invention is to use the screened bacterial strain as a fermentation strain for food fermentation to produce flavor and flavor substances.

The invention also aims to use the screened bacterial strain as a zymocyte for degrading ammonia nitrogen in water.

The initial ammonia nitrogen concentration of the water body is 50-600mg/L, and the ammonia nitrogen degradation efficiency of the water body is 47.78-88% after the water body is fermented by the strains for 24 hours.

The invention has the beneficial effects that:

the Pichia kudriavzevii strain provided by the invention is stable, high in growth speed, safe and simple in culture condition.

The Pichia kudriavzevii strain provided by the invention has completed a whole genome, and lays a foundation for researching the ammonia nitrogen degradation principle of the Pichia kudriavzevii source.

The Pichia kudriavzevii strain provided by the invention can produce fragrance by long-time fermentation, and lays a foundation for industrial application of fragrance-producing yeast.

The Pichia kudriavzevii provided by the invention is a Pichia kudriavzevii strain for treating ammonia nitrogen-containing wastewater, which is reported for the first time.

The Pichia kudriavzevii provided by the invention can treat ammonia nitrogen-containing wastewater by using ammonia nitrogen as a unique nitrogen source.

The Pichia kudriavzevii can utilize organic matters, is salt-resistant and heavy metal-resistant, can adapt to a wide pH range, and expands the environmental conditions for treating the wastewater containing ammonia nitrogen.

The strain is used for treating the wastewater containing ammonia nitrogen, the production process is environment-friendly, the operation is simple, no secondary pollution is caused, and the strain is very suitable for industrial large-scale production.

Drawings

FIG. 1 is a colony morphology of Pichia kudriavzevii HJ2 on YPD agar medium;

FIG. 2 is a cell morphology of Pichia kudriavzevii HJ2 under light microscope;

FIG. 3 is a cell morphology of Pichia kudriavzevii HJ2 under an electron microscope;

FIG. 4 is a plate diagram of ammonia nitrogen degradation by Pichia kudriavzevii HJ 2;

FIG. 5 is a physiological and biochemical characteristic diagram of Pichia kudriavzevii HJ2, wherein FIG. 5-a is a diagram showing growth effect of strains under different salinity conditions, FIG. 5-b is a diagram showing growth effect of strains under different pH conditions, FIG. 5-c is a diagram showing growth effect of strains under different culture temperatures, and FIG. 5-d is a diagram showing growth effect of strains under different heavy metal concentrations;

FIG. 6 is a chart of analysis of volatile flavor metabolites of the Pichia kudriavzevii strain HJ 2;

FIG. 7 is a genome-wide map of Pichia kudriavzevii strain HJ 2.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The materials and reagents used in the following examples are commercially available, unless otherwise specified.

In the present invention, the culture media used in the following examples:

YPD medium: 1% Yeast Extract, 2% Peptone (Peptone), 2% dextrose (glucose). Agar is added into the solid YPD culture medium for 1.5-2%. Sterilized at 115 ℃ for 15 minutes.

Carbon-free medium: (NH)₄)₂SO₄0.1％，K₂HPO₄0.7％，KH₂PO₄0.3%, trisodium citrate 0.05%, MgSO₄·7H₂O 0.01％。

Salinity culture medium: adding sodium chloride into YPD culture medium, and respectively preparing into salinity culture medium with sodium chloride concentration of 0-9%.

Carbon source assimilation medium: the carbon-free medium was supplemented with a sole carbon source to a final concentration of 2%. The carbon sources are glucose, sucrose, maltose, D-fructose, D-mannitol, D-galactose, L-arabinose, D-sorbitol, xylose, glycerol, soluble starch and lactose respectively, and the sterilization is carried out for 15 minutes at the temperature of 115 ℃.

Heavy metal ion tolerant media: in YPD liquid medium, heavy metal ion solutions were added to the respective concentrations of 0, 0.1, 0.5, 1, 3,5, 10, 25, 50 and 100mmol/L, and the pH was adjusted to 3.5.

Ammonia nitrogen degradation flat plate: glucose 0.5%, (NH)₄)2SO₄0.025％，NaCl 0.1％，K₂HPO₄·2H₂O 0.05％，MgSO₄·7H₂0.025% of O and 2% of agar.

Producing the aroma fermentation culture medium: carbon source (glucose) 2%, NaCl 0.05%, (NH)₄)₂SO₄0.2％，K₂HPO₄0.05％，KH₂PO₄0.05％，MgSO₄·7H₂O0.02%, sterilizing at 115 deg.C for 15 min.

Example 1 isolation and characterization of Pichia kudriavzevii HJ2

(1) Sampling from mangrove forest soil sediment in North sea city, Guangxi province, dissolving the sample with sterile water, shake culturing at 37 deg.C for 24 hr, and diluting with sterile distilled water to 10^-1、10^-2、10^-3、10^-4、10^-5Respectively take 10^-2～10^-5Respectively coating 100 mu L of the diluted bacterial liquid into YPD solid culture media, culturing for 2-3 days at 37 ℃, and carrying out plate streaking on single colonies which are milky, slightly convex, moist, irregular in edge, circular or elliptical and rough in surface for 2-3 times to obtain single colonies.

(2) Activated Pichia kudriavzevii HJ2

Inoculating the strain taken out from-80 deg.C, streaking in YPD solid culture medium, and culturing at 37 deg.C for 48 hr;

(3) the pichia kudriavzevii HJ2 was subjected to whole genome sequencing.

Pichia kudriavzevii HJ2 was sent to Nanning Pofil Biotech GmbH for whole genome sequencing.

Total number of Reads for whole genome sequencing is 343,523; total number of bases 1,109,084,396; the proportion of ambiguous bases was 0.001%, and the GC percentage content of total bases was 39.0%; the third generation sequencing result shows that the sequencing sequence has large length and no uncertain base. The total length of the sequence after the genome sequence is spliced is 10,911,289bp, and the GC percentage content is 38.16%; the prediction of ncRNA has two main categories of tRNA and rRNA; CAZy analyzes that there are 36 genes of glycoside hydrolase, 47 genes of glycosyl transferase, 6 genes of carbohydrate esterase and 7 genes of auxiliary active enzyme, and the genes do not contain polysaccharide lyase. As shown in tables 1-2.

TABLE 1 analysis of Whole genome sequencing results

Project	Detail	Project	Detail
				ScaffoldTotal(Mb)	10.91	tRNAs	206
ContigTotal(Mb)	10.91	rRNAs	20
				Scaffolds	19	RepeatsSize(bp)	65
Contigs	19	genome％GC	69.2％
				ScaffoldN50(Mb)	1.36	totalgenes	5087
Contig50(kb)	1364

TABLE 2 statistics of CAZy analysis results

(4) Molecular biological identification of Pichia kudriavzevii HJ2

Using the DNA of HJ2 strain obtained by separation and purification as a target fragment, and adopting a fungus universal primer to amplify 26SrDNA gene, wherein the primer is as follows:

NL1：5’-GCATATCAATAGCGGAGGAAAAG-3’

NL4：5’-GGTCCGTGTTTCAAGACGG-3’

amplifying 26S rRNA, sending the rRNA to Shanghai worker for sequencing, removing primers at two ends of a sequence obtained by sequencing to obtain a 601bp sequence, carrying out BLAST comparison on the sequence group on NBCI, and screening a sequence with higher homology, wherein the result shows that the sequence has higher homology (more than 99%) with 26S rDNA sequence of Pichia pastoris (Pichia kudriavzevi) in a library.

(5) Morphological feature observation of Pichia kudriavzevii HJ2

Pichia kudriavzevii HJ2 was cultured in YPD solid medium at 37 ℃ for 48 hours, and observed to be milky white, slightly convex, moist, irregular, round or oval in edge, rough in surface, and fast in growth, as shown in FIG. 1.

And (3) selecting a single colony on a glass slide, dropwise adding a drop of distilled water, covering the glass slide, observing the single colony by using a low-power lens, and then observing the characteristics of the single colony by using a 100-power lens, wherein the bacterial cell is oval and one end of the bacterial cell germinates. The form of the cells was observed under an electron microscope as shown in FIG. 2, and as shown in FIG. 3.

(6) Identification of physiological and biochemical characteristics of Pichia kudriavzevii HJ2

1) Preparing a seed solution: activated single colonies were picked up and cultured in YPD liquid medium at 37 ℃ at 200 rpm for 12 hours.

2) Culturing under different salinity conditions: the strain is 2% of the volume percentage of the salinity culture medium, and the strain can grow under the condition of 0-9% by culturing at 37 ℃ and 200 rpm for 24 hours, as shown in figure 5 (a).

3) Culturing under different pH conditions: the pH of the liquid YPD medium was adjusted to 1.5, 3.0, 7.0 and 9.0, respectively, using HCl and NaOH solutions as buffers, and 2% of the volume percentage of the YPD liquid culture medium as inoculum size, and the medium was cultured at 37 ℃ and 200 rpm for 24 hours with a pH growth range Ph2.0-9.0, as shown in FIG. 5 (b).

4) Culturing under different temperature conditions: the YPD liquid culture medium was cultured at 15 ℃, 20 ℃, 25 ℃, 30 ℃, 37 ℃, 45 ℃, 48 ℃, 50 ℃, 55 ℃ and 200 rpm for 24 hours with an optimum growth temperature of 30-37 ℃ using 2% by volume of the YPD liquid culture medium as the inoculum size, as shown in FIG. 5 (c).

5) Carbon source assimilation experiment: 2% of the volume percentage of the carbon source assimilation culture substrate was used as the inoculum size, and the culture was carried out at 37 ℃ and 200 rpm for 12 hours, and the results are shown in the following Table 3:

TABLE 3 carbon source assimilation

Carbon source	Results	Carbon source	Results	Carbon source	Results
						Glucose	+	D-mannitol	+	Xylose	+
Sucrose	+	D-galactose	+	Lactose	-
						Maltose	+	L-arabinose	+	Soluble starch	+
D-fructose	+	D-sorbitol	+	Glycerol	+

+ represents an assimilable carbon source and-represents an assimilable carbon source.

6) And (3) heavy metal ion tolerance culture: 2 percent of the volume percentage of the heavy metal ion tolerant culture medium is used as the inoculation amount, the heavy metal ion tolerant culture medium can grow under the environment of heavy metals (Cr, Cu, Mn, Co, Ni and Zn) with the pH value of 3.5 after being cultured for 24 hours at the speed of 200 r/min, and has strong tolerance to the heavy metal environment with the concentration of 24ppm, 640ppm, 5494ppm, 118ppm, 295ppm and 654ppm respectively, as shown in the table 4 and the figure 5 (d).

TABLE 4 HJ2 heavy metal tolerance and integrated wastewater discharge standard

Species of	GB25467-2012 comprehensive sewage discharge standard	Ion concentration for HJ2 growth
			Cr	0.1ppm	24ppm
Cu	2ppm	640ppm
			Mn	5ppm	5494ppm
Co	-	118ppm
			Ni	1ppm	295ppm
Zn	5ppm	654ppm

7) And (3) degrading ammonia nitrogen and identifying: inoculating the activated Pichia kudriavzevii HJ2 single colony in an ammonia nitrogen degradation plate; incubated at 37 ℃ for 24 hours. Colonies were observed to outgrow, i.e., pichia kudriavzevii HJ2 degraded ammonia nitrogen, as shown in fig. 4.

The strain is identified as a strain of Pichia kudriavzevi, namely Pichia kudriavzevi HJ2, by combining the molecular biology, strain morphology and physiological and biochemical characteristics, and is preserved in China general microbiological culture Collection center (CGMCC) of China micro-organism culture Collection, China Committee for culture Collection, China school No. 3 of West Lu 1 of North Chen of the sunward area in Beijing on the year of 2020 and on the year of 01 month, wherein the preservation number is CGMCC No. 19897.

Example 2 headspace gas chromatography detection of volatile aroma substances

(1) Fermentation flavor determination

Taking YPD culture medium and glucose culture medium (aroma-producing fermentation culture medium), taking 2% of volume percentage as inoculum size, culturing at 37 deg.C for 12 hr, 1 day, 3 days, 5 days, and 7 days, smelling the flavor of liquid fermentation broth, and the liquid fermentation broth has special fragrance such as sweet aroma, fruit acid, etc.

(2) Special flavour metabolite detection

Samples of pichia kudriavzevii HJ2 were fermented for 12 hours, 1 day, 3 days, 5 days, and 7 days with YPD medium and glucose medium, respectively, and analyzed with a headspace gas chromatograph. GC conditions were as follows: separating by using a DB-5 MS elastic quartz capillary chromatographic column SH-Rxi-5Sil MS chromatographic column (30m multiplied by 0.25mm multiplied by 0.25 mu m); the temperature programming condition is that the initial temperature is 40 ℃, the temperature is raised to 120 ℃ at the speed of 3 ℃/min, and the temperature is kept for 2 min; heating to 250 deg.c at 10 deg.c/min for 3 min; heating to 300 ℃ at a speed of 15 ℃/minute; hold for 2 minutes. The carrier gas was high purity helium (1.0 mL/min) and was injected without split flow.

MS conditions: the mass spectrum adopts an electron bombardment ionization source; electron energy 70 eV; the ion source temperature is 230 ℃; the scanning range is 35-350 m/z.

The amounts of the types of the special flavor metabolites obtained in the glucose fermentation medium for 12 hours, 1 day, 3 days, 5 days and 7 days were 55.32%, 62.00%, 40.86%, 50.71% and 46.15%, respectively, and the contents of the special flavor metabolites were 32.46%, 60.35%, 68.27%, 62.07% and 90.22%, respectively.

The aroma metabolites are esters, aldehydes, ketones, furans, acids and alcohols, and the main aroma components are isoamyl acetate, 4-methylbenzaldehyde, phenethyl alcohol, isovaleraldehyde, phenethyl acetate, diisobutyl phthalate, octanoic acid, (E) -3-decenoic acid, 2, 6-bis (tert-butyl) phenol, bis (2-methylpropyl) 1, 2-phthalate, farnesol, coumarin, indole, levogyration, beta-dihydro-beta-ionone, linolenic acid, lupinene, nerolidol, alpha-caryophyllene and the like. As shown in tables 5, 6, and 6.

TABLE 5 analysis of specific fragrance substances

Incubation time	Species of particular fragrant substances	Content of specific aromatic substances
			12 hours	55.32％	32.46％
1 day	62.00％	60.35％
			3 days	40.86％	68.27％
5 days	50.71％	62.07％
			7 days	46.15％	90.22％

TABLE 6 main Special aroma components of YPD culture medium

Example 5 determination of Ammonia Nitrogen degradation efficiency by salicylic acid colorimetric method

(1) Preparation of ammonia nitrogen culture medium

Culture medium: 20g of glucose, 0.25g of ammonium sulfate, 1.0g of sodium chloride, 0.5g of dipotassium phosphate, 0.25g of heptahydrate and magnesium sulfate, 1mL of trace element solution and 1L of distilled water. 100mL of the prepared solution is taken and distributed in a 250mL conical flask, and the conical flask is sterilized for 15 minutes at 115 ℃, and the ammonia nitrogen content in the non-inoculated ammonia nitrogen culture medium is 50 mg/L.

Solution of trace elements: 10.0g of EDTA, 1.2g of zinc sulfate, 1.5g of calcium chloride, 1.0g of manganese chloride tetrahydrate, 2.0g of ferrous sulfate, 1.0g of ammonium molybdate, 1.0g of pentahydrate and copper sulfate, 1.0g of cobalt chloride and 1LpH 7.2.2-7.4 of distilled water.

The initial ammonia nitrogen concentration of the culture medium is 50mg/L, and the content of ammonium sulfate is adjusted according to the proportion, so that the initial ammonia nitrogen concentration of the culture medium is 50mg/L, 150mg/L, 300mg/L, 400mg/L and 600mg/L in sequence.

(2) 2% seed solution (log phase) was added and shake-cultured at 37 ℃ for 24 hours at 200 rpm. Taking the 24-hour fermentation liquor, centrifuging at 12000 rpm for 10 minutes, measuring the ammonia nitrogen content in the supernatant, and calculating the ammonia nitrogen degradation efficiency.

(3) The test Saccharomyces curvatus was fermented and the assimilation of ammonia nitrogen was determined. The measurement result shows that the assimilation efficiency of the Saccharomyces curvatus HJ2 for ammonia nitrogen can reach 47.78% -88% within 24 hours, as shown in Table 7.

TABLE 7 measurement results of ammonia nitrogen assimilation rate

(4) Database comparison

The HJ2 whole genome sequences were compared within the database of related nitrogen cycles (NCycDB). Glutamate Dehydrogenase (GDH) was aligned to 2 genes, accounting for 3.90% of all aligned nitrogen circulating genes; glutamate Synthetase (GS) was aligned to 9 genes, accounting for 17.65% of all aligned nitrogen-circulating genes; glutamine synthetase (glnA) was aligned to 8 genes, accounting for 15.70% of all aligned nitrogen-circulating genes. The assimilation of HJ2 to ammonia mainly depends on the combined action of Glutamate Dehydrogenase (GDH) and Glutamine Synthetase (GS), wherein the glutamate dehydrogenase catalyzes the oxidative deamination of glutamate into alpha-homoglutaric acid through NAD or NADP accessory factors, and the product of the process is ammonia (the reference is the expression analysis of the glutamate dehydrogenase gene of Fenneropenaeus chinensis under the stress of ammonia nitrogen). The glutamine synthetase reduces the concentration of ammonia nitrogen in the body by converting excessive ammonia and glutamic acid in microorganisms into glutamine (reference: cloning of glutamine synthetase gene of Chenjinsong-Penaeus monodon and influence of ammonia nitrogen stress on the time-space expression thereof). Therefore, the HJ2 can be further confirmed to have a good ammonia nitrogen degradation function.

And (4) conclusion: the embodiment shows that the strain can be applied to high-concentration ammonia nitrogen water body treatment and the like, effectively treats sewage, does not produce subsequent pollution, is high in speed, is environment-friendly and sustainable, and has remarkable economic benefit.

Sequence listing

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Claims

1. The aroma-producing Pichia de-rienzi yeast for degrading ammonia nitrogen is named as Pichia de-rienzi yeast HJ2, and is deposited in China general microbiological culture Collection center in 2020, 06-01.01.numbered CGMCC No. 19897.

2. The use of pichia pastoris, ldiazianum, with characteristics of ammonia nitrogen degradation, as a fermentation strain for food fermentation to produce aroma and flavor substances.

3. The application of pichia kudriavzevii (vi) as a fermentation strain for degrading ammonia nitrogen in food fermentation for producing fragrance and increasing flavor substances is characterized in that the application comprises the step of inoculating the pichia kudriavzevii into a culture medium for fermentation, the fermentation temperature is 15-48 ℃, the fermentation pH is 2.0-9.0, and the weight content of sodium chloride in the culture medium is 0-9%.

4. The application of Pichia pastoris of producing fragrance and library of producing ammonia nitrogen as fermentation strain for improving fragrance and flavor in food fermentation according to claim 3, wherein the fermentation temperature is 37 ℃ and the fermentation pH is 3.0-8.0.

5. The application of pichia pastoris, ldireagkis, as a fermentation strain, for producing fragrance and flavor substances in food fermentation, for degrading ammonia nitrogen according to claim 3, wherein the fermentation time is 12 to 168 hours.

6. The application of the Pichia shibara De-Azi with ammonia nitrogen degradation as a fermentation strain in producing aroma and increasing flavor substances in food fermentation is characterized in that the carbon source contained in the culture medium is any one of glucose, sucrose, maltose, D-fructose, D-mannitol, D-galactose, L-arabinose, D-sorbitol, xylose, glycerol and soluble starch.

7. The application of pichia pastoris, i.e., pichia pastoris, with ammonia nitrogen degradation, as a fermentation strain for food fermentation to produce fragrance and flavor substances, wherein the number of types of fragrance metabolites obtained by fermentation is 40-62%, and the content of fragrance substances is 32-90.22%.

8. The use of pichia pastoris, cinnamomi kudriavzevii, capable of degrading ammonia nitrogen, as a fermentation strain for producing aroma and flavor substances in food fermentation, wherein the aroma metabolite species include esters, aldehydes, ketones, furans, acids, alcohols, and the aroma components include isoamyl acetate, 4-methylbenzaldehyde, phenethyl alcohol, isovaleraldehyde, phenethyl acetate, diisobutyl phthalate, octanoic acid, (E) -3-decenoic acid, 2, 6-bis (tert-butyl) phenol, bis (2-methylpropyl) 1, 2-phthalate, farnesol, coumarin, indole, levorotatory, β -dihydro- β -ionone, linolenic acid, lupalene, nerolidol, α -caryophyllene.

9. The use of pichia pastoris, ldiazianum, for degrading ammonia nitrogen, as defined in claim 1, as a fermentation strain for degrading ammonia nitrogen in water.

10. The application of pichia pastoris of shewanese doriazivi for degrading ammonia nitrogen in a water body according to claim 9, wherein the initial ammonia nitrogen concentration of the water body is 50-600mg/L, and the ammonia nitrogen degradation efficiency of the water body is 47.78-88% after the water body is fermented by a strain for 24 hours.

11. The use of pichia pastoris, ldiazevii, for degrading ammonia nitrogen, for tolerating heavy metals Cr, Cu, Mn, Co, Ni, Zn, as defined in claim 1, wherein the concentrations of heavy metals Cr, Cu, Mn, Co, Ni, Zn are 0-24ppm, 0-640ppm, 0-5494ppm, 0-118ppm, 0-295ppm, 0-654ppm, respectively.