CN110655196A - Method for treating nitrite nitrogen wastewater by using heterotrophic nitrification-aerobic denitrification pseudomonas strain - Google Patents

Abstract

The invention discloses a method for treating nitrite nitrogen wastewater by using a heterotrophic nitrification-aerobic denitrification pseudomonas strain, wherein the nitrate nitrogen wastewater contains organic acid sodium, and the C/N ratio of the nitrite nitrogen wastewater is 8-50, and the method comprises the following steps: inoculating the heterotrophic nitrification-aerobic denitrification pseudomonas seed liquid into nitrite nitrogen wastewater, and carrying out oscillation reaction for 6-24 h under the conditions that the temperature is 25-40 ℃ and the rotating speed is 90-180 rpm, so as to complete the treatment of the nitrite nitrogen wastewater; the strain is pseudomonas LJ9, is preserved in Guangdong province microorganism strain preservation center, and has the preservation number of GDMCC NO: 60339. the strain LJ9 has the capability of tolerating higher C/N and can tolerate nitrite nitrogen with higher concentration, so the method has great application value in the aspect of denitrification of wastewater with high C/N and high nitrite nitrogen.

Description

Method for treating nitrite nitrogen wastewater by using heterotrophic nitrification-aerobic denitrification pseudomonas strain

Technical Field

The invention relates to the technical field of microorganisms, in particular to a method for treating nitrite nitrogen wastewater by utilizing a heterotrophic nitrification-aerobic denitrification pseudomonas strain.

Background

In recent years, the problem of water pollution is increasingly severe, nitrogen is an important pollution factor in water, excessive nitrogen flows into water, water eutrophication is easily caused, insufficient dissolved oxygen in water is caused, water quality is deteriorated, the ecological environment of the water is changed, the survival of aquatic organisms is damaged, and the pollution to the environment and the health of human beings is extremely large. Nitrogen exists in water in two forms of organic nitrogen and inorganic nitrogen, and the main source of the organic nitrogen is domestic sewage, agricultural nitrogen fertilizer and industrial wastewater; inorganic nitrogen is decomposed and converted by microorganisms and mainly comprises ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, wherein the nitrate nitrogen can be converted into the nitrite nitrogen in a human body, and the nitrite is excessively taken, so that the methemoglobinemia is induced, and the suffocation is caused in serious cases. Nitrate-containing foods and water are more dangerous to infants and can lead to blue infants.

The problem of nitrogen pollution of the environmental water body is solved. Because the cost of the biological denitrification treatment is low,and has no secondary pollution to the environment, thus becoming one of the most common sewage denitrification methods at present and being valued at home and abroad. The conventional biological denitrification process is mainly based on Nitrification (NH) of autotrophic microorganisms under aerobic conditions₄ ⁺→NH₂OH→NO₂ ^-→NO₃ ^-) And denitrification of heterotrophic microorganisms under anaerobic conditions (NO)₃ ^-→NO₂ ^-→NO→N₂O→N₂). In the technology, autotrophic bacteria and heterotrophic bacteria need to be utilized, the autotrophic bacteria grow slowly by taking an inorganic carbon source as a raw material and have strong environmental sensitivity, and the heterotrophic bacteria need a large amount of organic matters for growth and have high energy consumption. Because of different nutrient substances and growth conditions in the nitrification and denitrification processes, large-scale equipment is needed, the time is long, and the theoretical denitrification efficiency is low. 1983, Robertson and the like firstly discover heterotrophic nitrification-Aerobic Denitrification bacteria Paracoccus, and put forward the concept of heterotrophic nitrification-Aerobic Denitrification (HN-AD) process, so that the traditional biological Denitrification concept is broken through, and the novel biological Denitrification technology is developed along with the situation. Compared with the traditional ammoniation, nitrification and denitrification, the novel denitrification technology simplifies the process flow, and the nitrification and denitrification are synchronously carried out in the same reactor under the aerobic condition. The bacteria grow and propagate quickly, and have higher denitrification efficiency, simple operation and more economical and practical. Therefore, the development of novel efficient denitrification heterotrophic nitrification-aerobic denitrification bacteria separation screening and denitrification performance research becomes a research focus at present.

In recent years, successively learners have screened out some of HN-AD (NH)₄ ⁺→NH₂OH→NO₂ ^-→NO→N₂O→N₂) The strain of (1). The HN-AD bacteria have wide substrate utilization range, can utilize various inorganic nitrogen and organic nitrogen, and have strong environmental adaptability. However, the HN-AD strain disclosed at present has poor high carbon nitrogen ratio resistance and high-concentration nitrogen resistance, and restricts the practical application and popularization of the heterotrophic nitrification-aerobic denitrification biological nitrogen removal technology in water body pollution remediation.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for treating high-carbon-nitrogen-ratio and high-nitrite-nitrogen wastewater by using a heterotrophic nitrification-aerobic denitrification pseudomonas strain LJ 9.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for treating nitrite nitrogen wastewater by using heterotrophic nitrification-aerobic denitrification pseudomonas strains comprises the following steps of:

inoculating the heterotrophic nitrification-aerobic denitrification pseudomonas seed liquid into nitrite nitrogen wastewater, and carrying out oscillation reaction for 6-24 h under the conditions that the temperature is 25-40 ℃ and the rotating speed is 90-180 rpm, so as to complete the treatment of the nitrite nitrogen wastewater;

the strain is Pseudomonas LJ9(Pseudomonas indoxydans LJ9), is preserved in Guangdong province microbial strain preservation center, has the preservation time of 3 months and 21 days in 2018, and has the preservation number of GDMCC NO: 60339. .

In one embodiment, the concentration of the nitrite wastewater in the nitrite nitrogen wastewater is 100 mg-L^-1～300mg·L^-1。

In one embodiment, the C/N ratio of the nitrite nitrogen wastewater is 8-25, and the concentration of the nitrite wastewater is 100 mg.L^-1～150mg·L^-1。

In one embodiment, the inoculation amount of the heterotrophic nitrification-aerobic denitrification pseudomonas seed liquid is 3% -5%.

In one embodiment, the pH value of the ammonia nitrogen wastewater is 6-9.

In one embodiment, the sodium organic acid is one or more of sodium citrate, sodium succinate and sodium acetate.

In one embodiment, the heterotrophic nitrification-aerobic denitrification pseudomonas seed liquid is prepared by the following method:

inoculating the pseudomonas strain into an LB culture medium to be cultured to a logarithmic phase, centrifuging the obtained bacterial suspension to remove supernatant, washing, and adding sterile water to obtain pseudomonas seed liquid.

In one embodiment, the culturing process comprises: performing shaking culture at 25-35 ℃ and 100-180 rpm for 12-18 h.

In one embodiment, the LB medium comprises: bacteriological peptone 10 g.L^-15 g.L yeast extract^-1，NaCl 10g·L^-1，pH 7.0。

In one embodiment, sterile water is added to the bacterial liquid OD₆₀₀0.6 to 0.8.

Compared with the prior art, the invention has the advantages that:

the invention utilizes seed liquid obtained by culturing Pseudomonas strain LJ9 to treat ammonia nitrogen wastewater, Pseudomonas LJ9(Pseudomonas indoxydans LJ9) has the capability of tolerating high-concentration organic matters, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, and has excellent comprehensive performance. In the aerobic nitrosation process, the nitrogen concentration of nitrite is 100 mg.L^-1When the C/N is 8-25, the removal rate of nitrite nitrogen of the strain LJ9 is more than 55%, and the strain LJ9 can tolerate the nitrite nitrogen concentration as high as 300 mg.L^-1Compared with most aerobic denitrifying bacteria, the strain LJ9 has the capability of tolerating higher C/N, and the strain LJ9 can tolerate nitrite nitrogen with higher concentration, so that the problem of nitrite accumulation can be solved more effectively, the aerobic denitrification reaction process is not easy to be inhibited by nitrite, and new strain resources can be provided for treating high nitrite nitrogen wastewater with high C/N organic pollution.

In addition, the nitrate nitrogen concentration of the strain LJ9 in the aerobic denitrification process is 100 mg.L^-1C/N is 25-75, and the removal rate of nitrate nitrogen in 24 hours reaches more than 99 percent; compared with most aerobic denitrifying bacteria, the strain LJ9 has the capability of tolerating higher C/N, and the strain LJ9 can tolerate the nitrate nitrogen concentration as high as 600 mg.L^-1And the nitrate nitrogen removal rate can reach 90.05 percent in 24h, which shows that the LJ9 aerobic denitrification reaction process is less influenced by the inhibition of nitrate, and the aerobic denitrification reaction process is less influencedThe denitrification performance is superior to that of most aerobic denitrification strains reported at present. In the heterotrophic nitrification process, the strain LJ9 has a wide C/N adaptation range, and the ammonia nitrogen concentration is 100 mg.L^-1When the C/N is 8-120, the removal rate of the ammonia nitrogen of LJ9 reaches more than 80 percent, and the concentration of the ammonia nitrogen reaches 500 mg.L^-1The bacterial strain LJ9 can still remove ammonia nitrogen in the process, and has high tolerance to wastewater with high C/N and high ammonia nitrogen concentration and high ammonia nitrogen removal rate. In conclusion, the pseudomonas strain LJ9 has excellent comprehensive performance of treating nitrogen-polluted wastewater.

Pseudomonas LJ9(Pseudomonas indoxydans LJ9) deposited in the microbial cultures Collection center of Guangdong province (GDMCC for short), and having the address of No. 59, No. 5, of Michelia furiosaefolia Miyao No. 100, Guangdong province, with the collection number of GDMCC NO: 60339, and the preservation time is 3 months and 21 days in 2018.

Drawings

FIG. 1 is a colony morphology of Pseudomonas LJ9 used in the present invention.

FIG. 2 is a transmission electron micrograph of Pseudomonas LJ9 used in the present invention.

FIG. 3 shows the 16S rRNAPCR electrophoretogram of Pseudomonas LJ9 used in the present invention.

FIG. 4 shows homology analysis of Pseudomonas LJ9 used in the present invention.

FIG. 5 is the electrophoresis picture of the pseudomonas LJ9 functional gene napA adopted by the invention.

FIG. 6 is a graph showing growth and aerobic nitrosification denitrification curves of Pseudomonas LJ9 used in the present invention.

FIG. 7 shows the effect of different C/N on aerobic nitrous denitrification performance of Pseudomonas LJ9 used in the present invention.

FIG. 8 is a graph showing the effect of different nitrite nitrogen concentrations on the denitrification performance of Pseudomonas LJ9 used in the present invention.

FIG. 9 shows the effect of different C/N on the aerobic denitrification performance of Pseudomonas LJ9 used in the present invention.

FIG. 10 is a graph showing the effect of different nitrate nitrogen concentrations on the denitrification performance of Pseudomonas LJ9 used in the present invention.

FIG. 11 is a graph showing the effect of C/N on the heterotrophic nitrification denitrification performance of Pseudomonas LJ9 used in the present invention.

FIG. 12 shows the effect of different ammoniacal nitrogen concentrations on the growth of Pseudomonas LJ9 used in the present invention.

FIG. 13 shows the effect of different ammonia nitrogen concentrations on denitrification performance of Pseudomonas LJ9 used in the present invention.

Detailed Description

The invention is further described below with reference to specific preferred embodiments, without thereby limiting the scope of protection of the invention.

The heterotrophic nitrification-aerobic denitrification pseudomonas strain used in the following examples is deposited in the Guangdong province culture Collection (abbreviated as GDMCC) with the deposition number of GDMCC NO: 60339. The pseudomonas GDMCC NO: 60339 is named as Pseudomonas sp LJ9(Pseudomonas indoxydans LJ 9).

1) Separating, screening and purifying strains

The pseudomonas LJ9 of the embodiment is mainly obtained by screening through the following method:

1.1) enrichment culture: 50mL of activated sludge collected from Longjin sewage treatment plant in Longyan city, Fujian province is added into a 100mLDM culture medium, and 2-4 sterile glass beads are added into the culture medium at the same time, so as to break up a mud sample. The culture was continued at 30 ℃ for 5 days with shaking at 150 rpm. The above bacterial suspension was inoculated into a new DM medium at an inoculum size of 10%, and the culture was repeated twice in this manner to obtain an enriched bacterial suspension.

1.2) separation and purification: 0.2mL of the bacterial suspension was applied to a DM solid medium plate by 10-fold dilution and cultured in an incubator at 30 ℃. And (3) when a single colony grows out of the plate, selecting bacterial colonies with obviously different characteristics, and further carrying out plate streaking for many times to obtain a purified single colony.

1.3) primary screening: to determine the denitrifying power of the strains, the purified strains were cultured in KNO₃In DM solid medium plates, which are the sole nitrogen source, colonies that were able to grow on the plates were picked to complete the primary screening.

1.4) re-screening: the strains after primary screening are inoculated into a heterotrophic culture medium and a denitrification culture medium, and strains LJ9 with ammonia nitrogen and nitrate nitrogen degradation rates of 66% and 96% are screened out respectively.

1.5) preservation: the strain is preserved by adopting a method of slant preservation of beef extract peptone at 4 ℃ and low-temperature freezing preservation of glycerol suspension. The low-temperature freezing preservation method of the glycerol suspension comprises the following steps: mixing 50% glycerol distilled water with LB cultured strain suspension at a ratio of 1: 1, and storing at-80 deg.C with final concentration of glycerol of about 25%.

2) Strain identification

The identification process and the results of the pseudomonas LJ9 strain of the embodiment are as follows:

2.1) morphological identification

The isolated strains were observed for single colony morphology on solid plates. The shape of the thallus is observed by adopting a gram staining method and a transmission electron microscope.

Strain LJ9 was streaked on beef extract peptone agar medium for 48h as shown in FIG. 1: the large bacterial colony is round, neat, moist, smooth in surface, milky translucent, and small convex. Gram staining identifies the bacterium as gram negative. The strain morphology is shown in the transmission electron microscope image of FIG. 2: the cells were rod-shaped, 6X 12.5 μm in size, flagellated, and sporulated.

2.2) physiological and biochemical identification

The typical method for classifying bacteria in detail is based on the results of physiological and biochemical experiments of bacteria, and the physiological and biochemical characteristics of the strains in the experiments are identified according to the handbook for identifying common bacteria systems and the handbook for identifying Bergeming bacteria.

The strain LJ9 is subjected to various physiological and biochemical characteristics, and the results are shown in Table 1: LJ9 can survive under anaerobic and aerobic conditions, oxidase, catalase, starch hydrolysis, methyl red, grease hydrolysis, citrate, indole experiments and nitrate reduction experiments are positive, and glucose oxidation fermentation, lactose oxidation fermentation, urea hydrolysis, V.P reaction, gelatin liquefaction, acetic acid oxidation, pectin hydrolysis, hydrogen sulfide production and pyocyanin experiments are negative. The above results show that: LJ9 has similar physiological and biochemical characteristics to Pseudomonas and can secrete a lot of extracellular enzymes, oxidize sugars, produce a small amount of acid and do not ferment.

TABLE 1 physiological and biochemical Properties of Strain LJ9

Name of experiment	Results of the experiment	Name of experiment	Results of the experiment
				Oxidase enzyme	+	Nitrate reduction	+
Contact enzyme	+	Pyocyancin	-
				Oxidative fermentation of glucose	-	Indole experiments	+
Lactose oxidative fermentation	-	Milk with litmus	+
				Hydrolysis of urea	-	Production of hydrogen sulfide	-
Starch hydrolysis	+	Citric acid salt	+
				Methyl Red	+	Pectin hydrolysis	-
V.P	-	Oxidation of acetic acid	-
				Hydrolysis of fats and oils	+	Liquefaction of gelatin	-
With paraffin seal (without oxygen)	+	Paraffin-free seal (aerobic)	+

Note: in Table 3, "+" indicates positive, there was a reaction; "-" indicates negative, no such reaction. In the sugar fermentation experiment, "+" indicates no production of acid or gas; "-" indicates no acid or gas production.

2.3) molecular biological identification

The bacterium contains three kinds of ribosomal RNA, 5S rRNA, 16S rRNA and 23S rRNA. rRNA gene consists of conserved region and variable region, and has moderate relative molecular weight and high repeatability of sequence analysis. Therefore, it is now common to use the 16S rRNA gene as a target for sequence analysis to perform sequencing analysis of microorganisms for determining the species of bacteria.

2.3.1) extraction of bacterial genomic DNA

Extracting the bacterial genome DNA by using an Ezup column type bacterial genome DNA extraction kit and operating according to the specific operation steps of the kit.

Extracting strain DNA as PCR template for amplification. The primers are universal primers, namely upstream 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', downstream 1492R:5'-GGTTACCTTGTTACGACTT-3', reacted in a PCR amplificator.

2.3.2) amplification and sequencing of the 16S rRNA Gene

PCR amplification System (Total 25. mu.l): 10 XBuffer 2.5. mu.l, dNTP 0.5. mu.l, 27F 0.5. mu.l, 1492R 0.5. mu.l, template DNA 1. mu.l, Taq enzyme 0.25. mu.l, sterile water 19.75. mu.l.

The PCR reaction program settings are shown in table 2:

TABLE 2

Pre-denaturation	95℃	3min
			Denaturation of the material	95℃	45s
Annealing	55℃	45s
			Extension of	72℃	45s

In total 35 cycles were run, with the 35 th cycle being extended for 5min at 72 ℃. Storing at 4 ℃.

And after the PCR product is qualified through agarose gel electrophoresis detection, committing Shanghai Huada Gene company to complete sequence determination, and performing homology analysis on a sequencing result to obtain the phylogenetic position of the strain.

The result of agarose gel electrophoresis imaging of the PCR amplification product of the rRNA gene of strain LJ916S is shown in FIG. 3: the PCR product band is bright, the channel has no impurities, and the band position is above 1000bp, which indicates that the 16S rRNA gene target band is obtained. The 16S rRNA gene PCR product of strain LJ9 was purified and sequenced to obtain a sequence of 1362bp in length, and the results of homology analysis by BLAST are shown in fig. 4: the strain LJ9 has 99% similarity with the strain Pseudomonas indoloxydans and is named as Pseudomonas indoloxydans LJ 9.

2.3.3) amplification of NapA functional Gene

Nitrate reductases are classified into membrane-bound nitrate reductases (Nar) and periplasmic nitrate reductases (Nap), in which periplasmic nitrate reductase genes are preferentially expressed under aerobic conditions and function under both anaerobic and aerobic conditions. Using primer nap 1: 5 '-TCTGGACCATGG-GCTTCAACCA-3', nap2: 5'-ACGACGACCGGCCAGCGCAG-3', and carrying out amplification on a PCR instrument.

PCR amplification System (Total 25. mu.l): 10 XBuffer 2.5. mu.l, dNTP 0.5. mu.l, nap 10.5. mu.l, nap 20.5. mu.l, template DNA 1. mu.l, Taq enzyme 0.25. mu.l, sterile water to 25. mu.l.

The PCR reaction program settings are shown in table 3:

TABLE 3

Pre-denaturation	94℃	10min
			Denaturation of the material	94℃	1min
Annealing	55℃	1min
			Extension of	72℃	1.5min

A total of 30 cycles were run, with the 30 th cycle being extended for a further 10min at 72 ℃. Storing at 4 ℃.

And (3) carrying out 1% agarose gel electrophoresis detection on the PCR product, carrying out electrophoresis for 30min at a voltage of 220V, and carrying out imaging photographing by a gel imaging system.

The primer nap1/nap2 is used for amplifying the periplasmic nitrate reductase gene of the LJ9 strain, and an agarose gel electrophoresis chart is shown in FIG. 5: the target band is between 750bp and 1000 bp. The napA genes of periplasmic nitrate reductase subunits are all about 870bp, so that the primary judgment can be made that the LJ9 contains the napA gene, which indicates that the strain LJ9 has the aerobic denitrification function.

The pseudomonas LJ9 of the embodiment has good heterotrophic nitrification-aerobic denitrification performance, has high carbon nitrogen ratio resistance and high nitrate nitrogen and nitrite nitrogen resistance, and can be used for denitrification treatment of wastewater with high C/N ratio, high nitrate nitrogen and high nitrite nitrogen. When the method is used, the pseudomonas LJ9 is prepared into pseudomonas LJ9 seed liquid, and then the wastewater denitrification treatment is carried out, wherein the pseudomonas LJ9 seed liquid is prepared by adopting the following method:

activating the strain LJ9 in an LB culture medium, culturing to a logarithmic phase, taking a strain suspension at 4000rpm, centrifuging for 10min, removing a supernatant, washing with sterile water, centrifuging again to remove the supernatant, repeating for 2-3 times, and adjusting the OD600 of the strain to 0.6-0.8 with sterile water to be used as a seed solution.

The formula of the culture medium used is as follows:

denitrification enrichment medium (DM) (g.L)^-1)：Na₂HPO₄·7H₂O 7.9，KH₂PO₄1.5，NH₄Cl 0.3，MgSO₄·7H₂O0.1, sodium succinate (CH)₂COONa)₂·6H₂O 4.7，KNO₃0.3, 2 mL. L of trace element solution^-1，pH7.0-7.5。

Solution of trace elements (g.L)^-1)：EDTA50，ZnSO₄2.2，CaCl 5.5，MnCl₂·4H₂O 5.1，FeSO₄·7H₂O5.0, ammonium molybdate (NH)₄)6Mo₇O₂₄·4H₂O 1.1，CuSO₄·5H₂O 1.6，CoCI₂·6H₂O 1.6，pH 7.0。

Beef extract peptone medium (g.L)^-1): bacteriological peptone 10, beef extract 3, NaCl 10, pH 7.0-7.2.

LB liquid Medium (g.L)^-1): bacteriological peptone 10, yeast extract 5, NaCl 10, pH 7.0.

Heterotrophic nitrification medium (g.L)^-1)：(NH₄)2SO₄0.47, sodium succinate (CH)₂COONa)₂·6H₂O4.5, sodium citrate C₆H₅Na₃O₇·2H₂O3.27, Vickers salt solution 50 mL. L^-1，pH 7.0。

Vickers salt solution (g.L)^-1)：K₂HPO₄6.54，MgSO₄·7H₂O 2.5，NaCl 2.5，MnSO₄·H₂O 0.04，FeSO₄·7H₂O 0.05。

Denitrification Medium (g.L)^-1)：KNO₃0.72, sodium succinate (CH)₂COONa)₂·6H₂O4.5, (sodium citrate)

C₆H₅Na₃O₇·2H₂O3.27), vickers' salt solution 50mL, pH 7.0.

Nitrosation medium (g.L)^-1)：NaNO₂0.49, sodium succinate (CH)₂COONa)₂·6H₂O4.5 (sodium citrate C)₆H₅Na₃O₇·2H₂O3.27), vickers' salt solution 50mL, pH 7.0.

Note: the solid culture medium is prepared by adding 1.5-2% of agar into the liquid culture medium, and sterilizing all the culture media at 121 ℃ for 20min for later use.

The experimental instruments used in the examples are shown in table 4:

TABLE 4 Experimental instruments

The water quality testing and analyzing items and methods in the examples are shown in table 5:

TABLE 5 items and methods for Water quality testing analysis

Index (I)	Measurement method
		Ammoniacal Nitrogen (NH)4 +-N)	Spectrophotometry with Nas reagent
Nitrate Nitrogen (NO)3 --N)	Thymol spectrophotometry
		Nitrous Nitrogen (NO)2 --N)	N- (1-naphthyl) -ethylenediamine photometry
Total Nitrogen (TN)	Alkaline potassium persulfate digestion spectrophotometer
		Chemical Oxygen Demand (COD)	COD instrument digestion method
pH	UB-7 precision pH meter
		Absorbance OD of biomass of bacteria600	Measured at a wavelength of 600nm using an ultramicro spectrophotometer

Example 1:

a method for treating nitrite nitrogen wastewater by using the heterotrophic nitrification-aerobic denitrification pseudomonas strain LJ9 seed liquid comprises the following steps:

inoculating the seed liquid into triangular conical flasks (500 ml) containing 200ml of nitrosation medium at an inoculation amount of 3%, wherein the initial nitrite nitrogen concentration is 100 mg.L^-1The cells were incubated at 30 ℃ and 150rpm for 24 hours with shaking and were sampled every 6 hours for measurement. With NaNO₂Determination of NO in Medium as sole Nitrogen Source₃ ^--N accumulation, pH, NO₂ ^-Contents of N, TN and COD.

Research result of aerobic denitrification performance

The strain LJ9 takes sodium succinate as a carbon source and NaNO₂The growth curve and the nitrosation denitrification curve of 24h of culture are shown in figure 6 as the only nitrogen source: because of the growth lag phase of the thallus, the growth of 0-6h is obviously slower than that of 6-12h, and OD₆₀₀Grow exponentially in 6-12 h. OD at 12h₆₀₀A maximum of 0.347 is reached. OD 12-18h₆₀₀Decline, nutrient deficiency, metabolismThe product is accumulated, the growth of the strain is inhibited, and the death rate of the strain is increased. The time is 18-24h in the stable period. The removal rate of COD tends to be stable within 18-24h, and reaches 87.79% within 24 h. The pH increased with time from 7.37 to finally 7.98.

The nitrosation denitrification process has a small amount of NO₃ ^-Production of-N, 0.394 mg.L in 6h^-1NO of₃ ^-N, which subsequently degrades again, is only 0.163 mg. L for 24h^-1NO of₃ ^-Accumulation of-N, possibly NO if prolonged incubation is used₃ ^--accumulation of N. The strain LJ9 is proved to have aerobic denitrification capability. The removal rate of nitrite nitrogen and total nitrogen of 24h strain LJ9 is 68.89% and 40.62% respectively. Flavobacterium sp.FL211T pair NO screened by Ganmei et al₂ ^-The denitrification capability of N is low, and the nitrogen removal rate of the sodium nitrite reaches the maximum within 12 hours, but is only about 30 percent. The nitrosation denitrification performance of the strain LJ9 is superior to that of FL 211T.

Example 2:

sodium citrate is used as a carbon source, and the concentration of nitrite nitrogen or nitrate nitrogen is 100 mg.L^-1Inoculating the seed liquid into nitrite nitrogen culture medium or nitrate nitrogen culture medium with C/N of 8, 25, 50, 75, 100, 120 at 3%, respectively, culturing at 25 deg.C and 180rpm for 24 hr, sampling, and measuring NO₂ ^--N、NO₃ ^-Content of-N and TN and their pH values and OD₆₀₀。

Influence result of C/N on aerobic denitrification performance of LJ9

Strain LJ9 with NaNO₂As sole nitrogen source, starting NaNO₂The concentration is 100 mg.L^-1Cultured for 24h, under different C/N, the strain LJ9 is NO₂ ^-The case of N degradation is shown in FIG. 7: nitrite nitrogen, total nitrogen removal and OD with increasing C/N₆₀₀The pH tended to decrease. OD of Strain LJ9 at C/N8₆₀₀Reaches 0.373, the removal rates of nitrite nitrogen and total nitrogen are the highest, and are respectively 66.6 percent and 60.22 percent; when the C/N is higher than 50, the removal rate of nitrite nitrogen is lower than 20%.

The optimum C/N for denitrification of the aerobic denitrifying bacteria is 5-8, and less is more than 20. Compared with most aerobic denitrifying bacteria, the strain LJ9 has the capability of tolerating higher C/N, and can provide a new strain resource for treating high C/N organic pollution.

Example 3:

inoculating seed solution with 3% inoculum size of sodium citrate as carbon source and C/N of 8 into nitrous acid solution with nitrogen concentration of 100, 150, 200, 300, 400, 500 mg.L^-1In the nitrite nitrogen culture medium, after shaking culture at 25 ℃ and 180rpm for 24 hours, sampling and measuring NO₂ ^-Content of-N and TN and their pH values and OD₆₀₀。

Influence result of nitrite nitrogen content on LJ9 aerobic nitrosation performance

Strain LJ9 in different NO₂ ^-Culturing for 24h at-N concentration, NO₂ ^-The removal of-N is shown in FIG. 8: OD as nitrite nitrogen concentration increases₆₀₀Gradually decreases. When initial NO₂ ^-N concentration of 100 mg.L^-1And 150 mg. L^-1The bacterial strain LJ9 has high removal rate of nitrite nitrogen and total nitrogen, which is 54.64%, 45.84%, 48.46% and 44.60% respectively; initial nitrite nitrogen concentration of 200 mg.L^-1The removal rate of nitrite nitrogen is low; initial N concentration of 300 mg.L^-1When present, nitrite nitrogen is not degraded. Nitrite nitrogen concentrations of 100, 150, 200, 300 mg.L respectively^-1The removal rates of nitrite nitrogen are 2.28, 2.87, 1.28 and 0.00mg (L.h)^-1The total nitrogen removal rates are 2.02, 2.79, 0.44, 0.02mg (L.h)^-1。

Different kinds of bacteria have different tolerance degrees on nitrite nitrogen, when the concentration of the nitrite is too low, the nitrite cannot meet the requirement of a nitrogen source for the growth of the bacteria, and when the concentration is too high, the nitrite can generate toxic action on microorganisms to inhibit denitrification. For example: initial nitrite nitrogen concentration of 10 mg.L of Pseudomonas talasiY-11^-1And 48h, the removal rates of nitrite nitrogen and total nitrogen respectively reach 100% and 63.1%, and the removal rate of nitrite nitrogen is reduced along with the increase of concentration. Bacillus coagulans YX-6 with initial nitrite nitrogen concentration of 20 mg.L^-1Of nitriteThe nitrogen removal rate is close to 100 percent, and when the initial nitrite nitrogen concentration is increased to 100 mg.L^-1The removal rate of nitrite nitrogen is only 20%. Compared with the strain, the strain LJ9 can tolerate nitrite nitrogen with higher concentration, can more effectively solve the problem of nitrite accumulation, is not easy to be inhibited by nitrite in the aerobic denitrification reaction process, and can provide a strain source for high nitrite nitrogen wastewater treatment.

1. Study on aerobic denitrification of LJ9

1.1 influence of C/N on aerobic denitrification performance of LJ9

Sodium citrate is used as a carbon source, and the nitrogen concentration of nitrate is 100 mg.L^-1Inoculating the seed liquid into nitrate nitrogen culture medium with C/N of 8, 25, 50, 75, 100, 120 respectively at 3% inoculation amount, shake culturing at 25 deg.C and 180rpm for 24 hr with sodium citrate as carbon source, sampling, and measuring NO₂ ^--N、NO₃ ^-Content of-N and TN and their pH values and OD₆₀₀。

Influence result of C/N on aerobic denitrification performance of LJ9

Strain LJ9 with KNO₃As a sole nitrogen source, initial KNO₃The concentration is 100 mg.L^-1Cultured for 24h, under different C/N conditions, the strain LJ9 is NO-resistant₃ ^-The case of N degradation is shown in FIG. 9: when the C/N is 25-75, the removal rate of nitrate nitrogen reaches more than 99.5%, the removal rate of the total nitrogen is divided into 84.84%, 85.65% and 88.45%, when the C/N is 100, the removal rates of nitrate nitrogen and total nitrogen are slightly reduced to reach 98.68% and 80.01%, respectively, and the total C/N of the LJ9 aerobic denitrification is higher, so that the application range is larger.

Under different C/N conditions, different strains have different denitrification capabilities and a certain application range, and when the C/N reaches a certain value, the aerobic denitrification rate is in a stable state. The strain Bacillus cereus CZ1 easy to separate and study immediately takes potassium nitrate as nitrogen source, most preferably C/N is 6, and when C/N continues to increase, the denitrification efficiency is slightly reduced. The HN-AD bacterium Acinetobacter pittii A14 screened from Huangting forest has NO when C/N is 16₃ ^-Complete removal of-N, C/N8 or 20, NO₃ ^-the-N removal rate is reduced.In conclusion, the denitrification efficiency is affected due to insufficient carbon source caused by too low C/N, the denitrification performance of the thalli is easily reduced due to too high C/N, and the optimal C/N for denitrification of the aerobic denitrifying bacteria is 5-8, and less is more than 20. Compared with most aerobic denitrifying bacteria, the strain LJ9 has the capability of tolerating higher C/N, and can provide a new strain resource for treating high C/N organic pollution.

1.2 influence of nitrate nitrogen content on aerobic denitrification performance of LJ9

Inoculating the seed solution with sodium citrate as carbon source, C/N of 25, and 3% of inoculum size at nitric acid nitrogen concentrations of 100, 150, 200, 300, 400, and 500 mg.L^-1In the nitrate nitrogen medium, after shaking culture at 25 ℃ and 180rpm for 24 hours, sampling and measuring NO₃ ^-Content of-N and TN and their pH values and OD₆₀₀。

Influence result of nitrate nitrogen content on aerobic denitrification performance of LJ9

Strain LJ9 in different NO₃ ^-Culturing for 24h at-N concentration, NO₃ ^-The N removal case is shown in fig. 10: according to the strain LJ9, the biomass, the removal rate of nitrate nitrogen and the pH value are in a negative correlation with the concentration of nitrate nitrogen, namely, as the content of nitrate nitrogen is increased, the removal rate of biomass and nitrate nitrogen is reduced, and the pH value is reduced. When NO is present₃ ^-The concentration of N is 100, 150, 200, 300, 400, 500, 600 mg.L^-1The removal rates of nitrate nitrogen were 99.79%, 98.49%, 95.99%, 96.82%, 95.22%, 92.34%, and 90.05%, respectively, and the removal rates were 4.16, 6.16, 8.00, 12.10, 15.87, 19.24, and 22.51mg (L. h)^-1(ii) a The total nitrogen removal rates were 90.87%, 72.27%, 57.17%, 48.45%, 35.00%, 28.00%, 20.56%, respectively, with corresponding removal rates of 3.79, 4.83, 4.76, 6.06, 5.83, 5.14mg (L.h)^-1. Indicating that the strain LJ9 can tolerate 600 mg.L^-1The nitrate nitrogen concentration of (a) and the nitrate nitrogen removal rate is high, and if the incubation time is prolonged, the total nitrogen removal rate may continue to increase.

The growth, metabolism and denitrification processes of the strain both need enzyme participation, and the water is influenced by the over-high concentration of nitrate nitrogenThe substrate concentration of the environment is not favorable for the diffusion of enzymatic reaction; can be combined with an activator of enzyme to reduce the enzymatic reaction rate; meanwhile, excessive substrates are combined with enzyme molecules to form an intermediate product, so that the enzyme is inactivated, and the denitrification efficiency of the strain is inhibited. Different strains have different nitrate nitrogen loading capacities. Acinetobacter sp.HY2 is reported to have initial nitrogen concentrations of 100, 200, 300 mg.L^-1Then, the nitrate nitrogen removal rates for 48h were 85%, 70% and 47%, respectively; pseudomonaspaputida LY1 at an initial nitrogen concentration of 300 mg.L^-1In time, the nitrate nitrogen removal rate is only 46%; it is likely that higher nitrate nitrogen inhibits the action of denitrification enzymes. And the strain LJ9 can tolerate the nitrate nitrogen concentration as high as 600 mg.L^-1And the nitrate nitrogen removal rate can reach 90.05% in 24h, which shows that the influence of nitrate inhibition on the LJ9 aerobic denitrification reaction process is small, and the aerobic denitrification performance of the strain is superior to that of most aerobic denitrification strains reported at present. Can provide a strain source for the treatment of high-nitrate nitrogen wastewater.

2. Study on heterotrophic nitrification performance of LJ9

2.1 Effect of C/N on heterotrophic nitrification of LJ9

Ammonia nitrogen concentration 100 mg.L^-1Changing the content of carbon source, inoculating the seed liquid into heterotrophic nitrification culture medium with C/N ratio of 4, 8, 12, 16, 20, 25, 30, 35, 50, 75, 100, 120 and with 3% inoculation amount, respectively, using sodium citrate as carbon source, culturing at 30 deg.C and 150rpm for 24 hr by shaking culture, sampling and measuring NH₄ ⁺N, TN content and its pH and OD₆₀₀。

The results are shown in FIG. 11: the strain LJ9 has wide adaptation range to C/N, and the ammonia nitrogen removal rate of LJ9 reaches over 80 percent when the C/N is 8-120. Compared with most heterotrophic nitrification-aerobic denitrification bacteria reported at present, the growth and denitrification of LJ9 have high C/N resistance.

The high-concentration organic wastewater which is easy to be biodegraded can be divided into the following according to the property sources: industrial waste water, such as food industry waste water, etc. with farm and pasture products as raw materials; one is light industrial and metallurgical waste water, such as pharmaceutical waste water. Citric acid is the most important organic acid widely used in food, medicine, daily chemical and other industries. The embodiment shows that the strain LJ9 has high carbon-nitrogen resistance, so that the strain has good tolerance to high-concentration organic pollution, and has good denitrification effect by taking sodium citrate as a carbon source, which shows that the strain LJ9 has the advantages and potential of denitrification treatment of high-concentration citric acid wastewater.

2.2 influence of ammonia nitrogen concentration on heterotrophic nitrification performance of LJ9

Inoculating the seed liquid with ammonia nitrogen concentration of 100, 200, 300, 400, 500 mg.L respectively at an inoculation amount of 3%^-1In the heterotrophic nitrification culture medium, sodium citrate is used as a carbon source, shaking table is continuously cultured for 7 days at the C/N of 50, the temperature of 25 ℃ and the rpm of 180, sampling is carried out at regular time every day, and NH is measured₄ ⁺Content of-N, NO₂ ^--N and NO₃ ^-Cumulative amount of-N, and its pH and OD₆₀₀. Selection of Biomass (OD)₆₀₀) The highest day was used for measuring the COD and TN contents.

Results of influence of ammonia nitrogen concentration on heterotrophic nitrification performance of LJ9

The strain LJ9 was cultured for 7 days at different initial ammonia nitrogen concentrations, and the growth curve is shown in FIG. 12: initial ammonia nitrogen concentration of 100-400 mg.L^-1OD of LJ9₆₀₀With the increase of the ammonia nitrogen content, the growth lag phase is prolonged, and when the ammonia nitrogen concentration is 500 mg.L^-1When the strain LJ9 does not grow basically, the concentration is the limit of the tolerance of the ammonia nitrogen of LJ9, but the ammonia nitrogen can be removed by LJ9 at the concentration. The initial ammonia nitrogen concentration is 200-400 mg.L^-1In time, by day 7 of culture, LJ9 growth was already in stationary phase, OD₆₀₀All reach about 1.3, so the range of ammonia nitrogen concentration tolerance for the growth of LJ9 is wider.

The influence results of different initial ammonia nitrogen concentrations on the denitrification performance of the strain LJ9 are shown in FIG. 13: after 7 days of culture, the removal rates of ammonia nitrogen of LJ9 are 91.29%, 94.50%, 98.27%, 99.49% and 17.47%, respectively. At the initial ammonia nitrogen concentration of 100-^-1In the range, the ammonia nitrogen removal rate is improved along with the increase of the initial ammonia nitrogen concentration, and a small amount of nitrate nitrogen is accumulated; at the initial ammonia nitrogen concentration of 100-300 mg.L^-1The total nitrogen removal rate is increased along with the increase of the initial ammonia nitrogen concentration, but the initial ammonia nitrogen concentration is 400 mg.L^-1When the biomass, the total nitrogen and the COD are all reduced,OD₆₀₀the total nitrogen and COD removal rates are respectively reduced to 1.211 and 79.16 percent and 49.91 percent, which shows that the over-high nitrogen concentration can inhibit the growth and denitrification performance of the bacteria, and the highest ammonia nitrogen tolerance concentration of the strain LJ9 is about 500 mg.L^-1。

Different bacteria have different tolerance to high ammonia nitrogen. Heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas LY1 and Acinetobacter sp.HY2 with initial ammonia nitrogen concentration of 50 mg.L^-1The ammonia nitrogen removal efficiency is the highest, and is respectively 64% and 74%, and the ammonia nitrogen degradation rate is gradually reduced along with the increase of the ammonia nitrogen concentration. Sp YN3 of Acinetobacter sp.with the increase of initial ammonia nitrogen concentration, the lower the ammonia nitrogen removing ability, the initial ammonia nitrogen concentration increased to 200 mg.L within 1 day^-1In this case, denitrification reaction hardly occurred, and the initial ammonia nitrogen concentration was 150 mg.L^-1And the removal rate of the ammonia nitrogen is only 18.7 percent in 24 h. Compared with the strain, the initial ammonia nitrogen concentration is respectively 100, 200, 300, 400 and 500 mg.L^-1When the strain is cultured for 24 hours, the removal rates of ammonia nitrogen of the strain LJ9 are 87.86%, 41.55%, 9.37%, 8.69% and 10.38% respectively. Indicating that LJ9 has better tolerance to ammonia nitrogen concentration.

The above description is only for the preferred embodiment of the present application and should not be taken as limiting the present application in any way, and although the present application has been disclosed in the preferred embodiment, it is not intended to limit the present application, and those skilled in the art should understand that they can make various changes and modifications within the technical scope of the present application without departing from the scope of the present application, and therefore all the changes and modifications can be made within the technical scope of the present application.

Claims (10)

1. A method for treating nitrite nitrogen wastewater by using heterotrophic nitrification-aerobic denitrification pseudomonas strains is characterized in that the nitrite nitrogen wastewater contains organic acid sodium, the C/N ratio of the nitrite nitrogen wastewater is 8-50, and the method comprises the following steps:

inoculating the heterotrophic nitrification-aerobic denitrification pseudomonas seed liquid into nitrite nitrogen wastewater, and carrying out oscillation reaction for 6-24 h under the conditions that the temperature is 25-40 ℃ and the rotating speed is 90-180 rpm, so as to complete the treatment of the nitrite nitrogen wastewater;

the strain is Pseudomonas LJ9(Pseudomonas indoxydans LJ9), is preserved in Guangdong province microbial strain preservation center, has the preservation time of 3 months and 21 days in 2018, and has the preservation number of GDMCC NO: 60339.

2. the method of claim 1, wherein the nitrite nitrogen wastewater is treated by using a heterotrophic nitrification-aerobic denitrification pseudomonas strain, wherein the concentration of the nitrite wastewater in the nitrite nitrogen wastewater is 100 mg-L^-1～300mg·L^-1。

3. The method for treating nitrite nitrogen wastewater by using pseudomonas strain subjected to heterotrophic nitrification-aerobic denitrification as claimed in claim 2, wherein the C/N ratio of the nitrite nitrogen wastewater is 8-25, and the concentration of the nitrite wastewater is 100 mg-L^-1～150mg·L^-1。

4. The method for treating nitrite nitrogen wastewater by using the pseudomonas strain subjected to heterotrophic nitrification-aerobic denitrification as claimed in claim 3, wherein the inoculation amount of the pseudomonas seed solution subjected to heterotrophic nitrification-aerobic denitrification is 3% -5%.

5. The method for treating nitrite nitrogen wastewater by using the pseudomonas strain subjected to heterotrophic nitrification-aerobic denitrification as claimed in claim 4, wherein the pH of the ammonia nitrogen wastewater is 6-9.

6. The method for treating nitrite nitrogen wastewater by using a heterotrophic nitrification-aerobic denitrification pseudomonas strain according to claim 5, wherein the sodium organic acid is one or more of sodium citrate, sodium succinate and sodium acetate.

7. The method for treating nitrite nitrogen wastewater by using the heterotrophic nitrification-aerobic denitrification pseudomonas strain as claimed in any one of claims 1 to 6, wherein the heterotrophic nitrification-aerobic denitrification pseudomonas seed solution is prepared by the following method:

inoculating the pseudomonas strain into an LB culture medium to be cultured to a logarithmic phase, centrifuging the obtained bacterial suspension to remove supernatant, washing, and adding sterile water to obtain pseudomonas seed liquid.

8. The method for treating nitrite nitrogen wastewater by using heterotrophic nitrification-aerobic denitrification pseudomonas strain according to claim 7, wherein the culturing process comprises: performing shaking culture at 25-35 ℃ and 100-180 rpm for 12-18 h.

9. The method for treating nitrite nitrogen wastewater by using a heterotrophic nitrification-aerobic denitrification pseudomonas strain according to claim 8, wherein the LB medium comprises the following components: bacteriological peptone 10 g.L^-15 g.L yeast extract^-1，NaCl 10g·L^-1，pH 7.0。

10. The method of claim 9, wherein sterile water is added to the OD of the bacterial liquid₆₀₀0.6 to 0.8.

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