CN102676431A - Denitrifying bacteria and aquatic plant-microbe combined repair method using same - Google Patents

Abstract

The invention discloses a denitrifying bacterium and a method for combined restoration of water body plants and microbes. The denitrifying bacteria LR was preserved in the China Center for Type Culture Collection on May 3, 2011, and the strain preservation number is CCTCC NO: M2011159. A method for combined plant-microorganism restoration of nitrogen eutrophic water body, planting umbrella grass in nitrogen eutrophic water body, and inoculating denitrifying bacteria LR. In the present invention, the denitrifying bacteria LR is combined with the umbrella grass or the root secretion of the umbrella grass to repair the nitrogen eutrophication water body, and the bacterial strain LR can promote the repair of the nitrogen polluted water body by the umbrella grass. The method that the denitrifying bacteria LR provided by the present invention is used in combination with the umbrella grass or the root exudates of the umbrella grass to repair the nitrogen eutrophication water body is especially suitable for the treatment of the nitrogen eutrophication water body in nature.

Description

A kind of denitrifying bacteria and its method for water body plant-microorganism combined restoration

technical field

The invention belongs to the technical field of environmental (water body) pollution control, and relates to a denitrifying bacterium and a combined water plant-microorganism restoration method thereof, in particular to a denitrifying bacterium and a plant-microbe used for water body nitrogen eutrophication Microbial joint remediation method.

Background technique

With the development of social economy, river pollution is a problem faced by all countries in the world. Treatment of urban polluted water bodies is a basic measure to protect urban water resources and improve the ecological environment. However, the commonly used physical and chemical remediation methods are generally expensive in investment, complicated in equipment and not suitable for remediation and treatment of large-scale water pollution. However, microbial (denitrifying bacteria) remediation technology cannot be widely used due to the limitation of bacterial colony growth by the oxygen content in the water body and the types of available carbon sources. Up to now, most of the denitrifying bacteria isolated from the natural environment are facultative anaerobic bacteria, which use nitrate ion (NO ₃ ^- ) as the final electron acceptor for denitrification under anaerobic or hypoxic conditions; The denitrification is inhibited or even stopped under the condition of high oxygen concentration. The respiration of plant roots can reduce the oxygen content of water or soil in the root zone, and promote the denitrification of denitrifying bacteria. In addition, the type of carbon source available in the environment is an important factor affecting the survival of denitrifying bacteria. Plant roots can secrete a variety of carbon sources (sugars, amino acids, proteins, organic acids), which contain the nutrients necessary for the survival of denitrifying bacteria substance. A large number of studies have shown that the types and numbers of denitrifying bacteria located in the rhizosphere of plants are significantly higher than those in other areas. At present, a lot of research has been carried out on the remediation of water pollution by plants or microorganisms, but there are few studies on the treatment of nitrogen eutrophication in water bodies through the combined remediation technology of plants and microorganisms.

Contents of the invention

The object of the present invention is to provide a denitrifying bacterium used for nitrogen eutrophication treatment of water body.

Another object of the present invention is to provide the application of the denitrifying bacteria.

Another object of the present invention is to provide a combined plant-microbe restoration method for the denitrifying bacteria to be used for nitrogen eutrophication of water body.

The purpose of the present invention can be achieved through the following technical solutions:

A strain of denitrifying bacteria LR, classified as Pseudomonas sp. LR, was preserved in the China Center for Type Culture Collection on May 3, 2011, and the strain preservation number is CCTCC NO: M2011159. The Pseudomonas LR was isolated from the rhizosphere soil of eutrophic river plants in Nanjing City, Jiangsu Province. The main biological characteristics are: G ^- , rod-shaped bacteria. Indole test, glucose fermentation, lactose fermentation and starch hydrolysis test were negative, VP test was positive; antibiotic resistance test showed that LR strains were resistant to ampicillin, erythromycin, chloramphenicol, streptomycin; carbon source was available There are sodium citrate, sodium acetate, maltose, sodium succinate and starch, and non-available carbon sources are lactose and sucrose. The Genbank accession number of its 16S rDNA is JQ652571.

The application of the denitrifying bacteria LR with the preservation number CCTCC NO: M2011159 in the treatment of nitrogen eutrophication water pollution.

The application of denitrifying bacteria LR combined with Cyperus alternifolius L. with the preservation number of CCTCC NO: M2011159 in nitrogen eutrophication water pollution control.

A method for plant-microorganism joint restoration of nitrogen eutrophication water body, planting umbrella grass in the nitrogen eutrophication water body, inoculating the denitrification bacteria LR whose preservation number is CCTCC NO: M2011159; wherein each Plant 7-15 strains of umbrella grass per liter of water, and inoculate 10ml of denitrifying bacteria LR bacterial solution with a concentration of 106cFu/ml and a preservation number of CCTCC NO: M2011159 per liter of water.

A method for controlling nitrogen eutrophication water pollution. Adding umbrella grass root exudates to the nitrogen eutrophication water body, and inoculating denitrifying bacteria LR with the preservation number of CCTCC NO: M2011159, adding 50ml of umbrella grass root per liter of water body 10ml of denitrifying bacteria LR bacterial solution with the inoculation concentration of 10 ⁶ cFu/ml per liter of water body and the preservation number of CCTCC NO: M2011159. After inoculation, add umbrella grass root secretion every two days, each time Add 40-60ml, preferably 50ml per liter of water each time;

Among them, after the root system of umbrella grass is rinsed with deionized water, soak the root system of the plant in deionized water for 4 to 7 hours in a climate room with a relative humidity of 75-80%, a temperature of 25°C, and a light intensity of 3000Lx to 5000Lx, and use rotary evaporation concentration to 1/10/volume; the mass-volume ratio of the root system of the umbrella grass and the deionized water used to soak the root system is 1g: 10-30ml, preferably 1g: 20ml.

The standard of N element eutrophication liquid described in the present invention was announced by the International Health Organization (WHO) in 2004, and its standard is 11.3mg-N/l (Horing, H.and Chapman, D.: Nitrates and nitrites in drinking water: World HealthOrganization Drinking Water Series., IWA Publishing, London (2004).); that is, the liquid with nitrogen content greater than or equal to 11.3mg-N/l is called N eutrophication liquid, and the nitrogen content is greater than or equal to The water body equal to 11.3mg-N/l is called N eutrophication water body.

Beneficial effect:

The present invention separates and screens out a highly efficient denitrifying bacterium—Pseudomonas LR from soil in the rhizosphere zone of eutrophic river plants in Nanjing City, Jiangsu Province. The final degradation rate of the strain is more than 90%, and it also has a certain denitrification ability at a higher nitrogen level. It will be of great significance to expand the cultivation of this strain for the treatment of high NO ₂ ^- or high NO ₃ ^- wastewater.

On this basis, in the present invention, Pseudomonas LR is used in combination with the umbrella grass or the root exudates of the umbrella grass to repair nitrogen eutrophic water bodies, and the strain LR can promote the repair of nitrogen-polluted water bodies by the umbrella grass. The method that the Pseudomonas LR provided by the present invention is used in combination with the umbrella grass or the root exudates of the umbrella grass to repair the nitrogen eutrophication water body is especially suitable for the treatment of the nitrogen eutrophication water body in nature. The method significantly improves the survival rate of denitrifying bacteria in sewage, greatly improves the nitrogen removal rate of the restoration system, and saves the cost of adding carbon sources from external sources.

Description of drawings

Figure 1 Photos of strain LR gas production assay.

Figure 2 Colony photos of strain LR on BTB plate.

Fig. 3 Removal of nitrate nitrogen by strain LR.

Figure 4 Effects of different carbon sources on denitrification and denitrification by strains.

Figure 5 Effects of different nitrogen sources on the growth of strain LR.

Fig. 6 Effect of C/N on denitrification (A) and growth (B) of strain LR.

Fig. 7 The denitrification rate of different treatment groups using Hoagland nutrient solution as N eutrophication solution.

Figure 8. Nitrogen removal rates of different treatment groups using external sewage as N eutrophication liquid.

Fig. 9: Nitrogen removal experiment by using external sewage as the exogenous source of N eutrophication liquid and adding umbrella grass root exudates.

Biological Material Deposit Information

The denitrifying bacteria LR, classified as Pseudomonas LR (Pseudomonas sp. LR) is preserved in the China Center for Type Culture Collection, referred to as CCTCC, and the preservation address is Wuhan University, Wuhan, China, and the preservation date is May 3, 2011. The number is CCTCC NO: M2011159.

Detailed ways

Embodiment 1 strain screening and identification

1.1 Strain isolation and screening

The soil samples used in the test were collected from the roots of aquatic plants along several rivers and lakes in Nanjing, the bottom sludge of sewage treatment plants, and the soil of paddy fields in Lianyungang. Vigorously growing plants are selected, and after shaking off a large piece of surface soil, the remaining part is used as plant rhizosphere soil.

Weigh 10 g of the collected plant root soil sample, put it into 90 mL of sterile water containing glass beads, shake it for 20 min, and let it stand for 20-30 s to make a bacterial suspension. The bacterial suspension was transferred to DM medium with a 10% inoculum size and cultured at 28°C. Under sterile conditions, take 1mL of bacterial suspension and put it into a 9mL sterile water glass tube, mix well, and then make dilutions in various concentrations of 10 ^-2 , 10 ^-3 , 10 -4 , 10 ^{-5 ,} 10 ^-6 liquid.

Take 0.1mL of the above-mentioned dilutions of different concentrations and spread them on the BTB identification medium respectively, and set three replicates for each concentration. Cultivate at 28°C for 1-3 days, pick single colonies of different shapes with blue halos around them, and purify by streaking multiple times.

Pick the isolated strains, streak and purify them on the plate for many times, until no bacteria are found under the microscope, at this time, the strains can be considered as purified. The isolated strains grow well on the solid medium containing nitrate, and have potential denitrification characteristics, that is, the strains to be screened, and they are all inoculated to the slant medium for storage for later use.

The purified strains were respectively inserted into Giltay medium for re-screening, and cultured at 28°C for 2-14 days. Select the strains with fast gas production and more air bubbles, and further detect the content of nitrite nitrogen and total nitrogen in the medium. The strains with strong denitrification activity were screened out and kept on slant.

The cultivation and formula used in the above separation and screening process are:

Denitrification basal (DM) medium (Li Yan et al., 2008): KNO ₃ 2.0g, K ₂ HPO ₄ 1.0g, MgSO ₄ ·7H ₂ O 0.2g, sodium citrate 5g, distilled water 1000mL, pH 7.2.

Bromothymol blue (BTB) differential medium (TAKAYA et al., 2003): L-asparagine 1.0 g, KNO ₃ 1.0 g, KH ₂ PO ₄ 1.0 g, FeCl ₃ 6H ₂ O 0.05 g, CaCl ₂ 2H ₂ O 0.2g, MgSO ₄ 7H ₂ O 1.0g, agar 20g, 1% bromothymol blue 1mL, distilled water 1000mL, pH 7.0-7.3.

Giltay medium (Soil Microbiology Research Society, 1983):

A solution: KNO ₃ 2.0g, L-asparagine 1.0g, 1% bromothymol blue 5mL, distilled water 500mL;

Solution B: sodium citrate 8.5g, KH ₂ PO ₄ 1.0g, FeCl ₃ 6H ₂ O 0.05g, CaCl ₂ 2H ₂ O 0.2g, MgSO ₄ 7H ₂ O 1.0g, distilled water 500mL;

Mix the two solutions of A and B to adjust the pH value to 7.0-7.2.

Through enrichment separation, primary screening of BTB identification medium and re-screening of Gailty medium, a gas-producing denitrifying bacterium with the strongest denitrification activity was finally isolated from soil samples in the rhizosphere zone of eutrophic river plants in Nanjing City, Jiangsu Province , the strain was named LR, and LR was sent to the China Center for Type Culture Collection for preservation, and the strain preservation number was CCTCC NO: M2011159. Gas production photos are shown in Figure 1.

1.2 Determination of physiological and biochemical indicators of denitrifying bacteria

The identification of the strains was carried out with reference to the "Common Bacterial System Identification Manual" (Dong Xiuzhu et al., 2001) and "Bergey's Manual of Determinative Bacteriology" (Buchanan and Gibbons, 1986).

Strain LR is a Gram-negative bacillus, and forms white round, flat opaque colonies with slightly raised edges, dry and dull colonies on the BTB solid medium plate. Colony photos are shown in Figure 2.

Strain LR belongs to facultative anaerobic bacteria. Under anaerobic conditions, denitrifying bacteria use nitrate nitrogen as the final electron acceptor to complete nitrate metabolism. Gram-negative bacteria, VP-positive, can make litmus milk peptonization. Strain LR was able to utilize sucrose for fermentation. The specific results are shown in Table 1.

Table 1 Physiological and biochemical characteristics of strain LR

Note: "+" means the result is positive; "-" means the result is negative

1.3 Analysis and identification of 16S rRNA gene sequence of denitrifying bacteria

(1) Extraction of bacterial total DNA

(2) PCR amplification

The PCR reaction system preparation is shown in Table 2.

Table 2 Preparation of PCR reaction system

The PCR operation program was: 95°C pre-denaturation for 5 min, 94°C extension for 30 sec, 52°C annealing for 30 sec, 72°C extension for 1.5 min, 30 cycles, 72°C extension for 10 min, and 10°C incubation for 5 min.

The primers used for the PCR reaction of 16S rDNA are a pair of universal primers (Zhai Qian et al., 2007):

The forward primer is 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1);

The reverse primer is 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.2).

Store PCR products at 4°C.

(3) Determination and similarity analysis of 16Sr RNA gene sequence

After extraction and PCR amplification of strain LR DNA, a DNA fragment with a certain length of about 1.5kb was obtained. The PCR product was sent to a professional company for recovery, purification and sequence determination. The sequencing results showed that the 16S rRNA gene of strain LR had a total of 1436 bases Based on the similarity of the 16S rRNA gene sequence of related bacteria in GenBank (http://www.ncbi.nlm.nih.gov) and its RDP (http://rdp.cme.msu.edu) through the BLAST program Comparison. According to the sequencing results, using clustalxl.83, Mega4 and other related software to compare and analyze the homologous sequences found in GenBank and RDP databases, it was found that the strain LR had the highest homology with Pseudomonas. Biochemical characteristics analysis and 16S rRNA gene homology sequence comparison identified LR as Pseudomona sp.

Example 2 Research on the denitrification characteristics of Pseudomonas LR

2.1 Denitrification capacity determination

Take 8 mL of the bacterial suspension cultured at 28°C for 36 hours and inoculate it into 500 mL of sterilized DM culture solution (packed in a triangular bottle), mix evenly, and then divide into sterile test tubes, 10 mL each, and culture at 28°C for 6 hours and 12 hours respectively. , 18h, 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h, and 72h, take samples, take them out and store them in a refrigerator at 4°C, take out 3 tubes each time, and measure the removal rate of nitrate nitrogen after all are taken out.

When the bacteria are inoculated into the fresh DM medium, the bacteria do not grow and multiply immediately, but need to be adjusted and adapted for a period of time to synthesize a variety of enzymes and improve the enzyme system in the body and other components of the cell. A shorter delay period indicates a stronger ability to adapt to the environment. Different bacteria have different growth characteristics, and the denitrification ability of bacteria in different periods can be reflected by measuring the change of nitrate nitrogen.

The denitrification capacity of LR is shown in Figure 3. It can be seen from the figure that the whole growth cycle of the strain LR is about 3 days, the logarithmic growth phase is longer, and it is in the stable growth phase at about 54 hours. Its denitrification mainly occurs in the logarithmic growth phase (12-54h) of its growth, and lasts for a long time.

2.2 Carbon source and nitrogen source test

Carbon source test: 0.5% (mass fraction) of sodium acetate, sodium citrate, sodium succinate, sucrose, lactose, starch, and maltose were added to the denitrification medium without carbon source as the only carbon source, and other ingredients constant. Take 10 mL of each prepared medium and put it into a test tube for sterilization, and then inoculate the activated cells of Pseudomonas LR in the logarithmic phase with 10% of the inoculum. Cultivate statically at a constant temperature of 28°C, observe and record the gas production phenomenon, take samples to detect TN after 2 days, and analyze the influence of different carbon sources on the denitrification ability of the strain.

It can be seen from Figure 4 that the three strains all have a relatively broad spectrum of carbon sources and can use a variety of carbon sources. When culturing for 2 days, when the strain LR uses sodium citrate as the carbon source, the nitrogen removal rate is 70% after two days. %above.

Nitrogen source test: With sodium citrate as the carbon source, 0.1% (mass fraction) of potassium nitrate, sodium nitrite, acetamide, ammonium chloride, and urea were added to the denitrification basal medium without nitrogen source as the only As carbon source, inoculate the logarithmic phase Pseudomonas LR activated cells after killing the medium, which can eliminate the influence of the nitrogen source brought into the liquid. Culture at constant temperature at 28°C for 2 days, and measure the change of OD ₆₀₀ to indicate the growth status of the bacteria under different nitrogen source conditions.

It can be seen from Figure 5 that for the five different forms of nitrogen sources used in the experiment, the strain LR showed certain adaptability, and the biomass achieved by using potassium nitrate was the largest. Some studies have shown that due to the toxic effect of nitrite, some strains cannot grow using nitrite (Zhang Guangya et al., 2005). The diversity shows that it can adapt to the water quality of different types of nitrogen pollution, which is conducive to its popularization and application.

2.3 Effect of carbon-nitrogen ratio on denitrification

Taking KNO ₃ as the sole nitrogen source and sodium citrate as the carbon and energy source, the effects of different C/N (molar ratio) on the growth of denitrifying bacteria and nitrogen removal ability were investigated. Add 200mL of sterilized culture medium to a 250mL Erlenmeyer flask, inoculate the inoculum of pre-culture ld at a volume ratio of 1%, seal it with a rubber stopper (deep closed culture) and put it into a shaking incubator (140r min ^-1 ) at 28°C Constant temperature culture. The initial pH value was adjusted to 7.0, and the TN content was maintained at lgL ^-1 . By adjusting the amount of sodium citrate, the C/N ratios were 1, 3, 5, 7, 10 and 15, respectively. Sampling was taken regularly to detect OD ₆₀₀ and TN, and the influence of C/N on the denitrification process was analyzed.

Figure 6(A) and (B) respectively reflect the change of nitrogen purification efficiency and biomass of strain LR under different C/N conditions. With the increase of C/N, the biomass and total nitrogen removal rate of strain LR showed an upward trend. When the C/N was 7, the biomass of the strain LR was the largest, and the percentage of nitrogen purification was also the highest. When C/N>10, both biomass and removal percentage began to decrease.

Example 3 Using Hoagland nutrient solution as N eutrophication liquid to implement plant-microbe joint restoration

Collection method of umbrella grass root exudates: Weigh about 10g of umbrella grass, rinse the root system of umbrella grass several times with deionized water, then use filter paper to dry the surface water, put the plant into 200mL distilled water, at a temperature of ( 25±l) ℃, light intensity of 4000Lx, relative humidity of 75-80% in the artificial climate chamber for about 6 hours, after taking out the plants, filter the root exudate solution immediately, use a rotary evaporator to concentrate to 20ml, -20 Store at ℃ for later use, or directly save for later use.

Choose the same growth, in the Hoagland nutrient solution (Ca(NO ₃ ) ₂ , 945mg/l; KNO ₃ , 607mg/l; (NH ₄ ) ₃ PO ₄ , 115mg/l; MgSO ₄ , 493mg/l; 2.5ml/ l Iron solution: FeSO ₄ ·7H ₂ O, 5.56g/l; Na ₂ ·EDTA, 7.46g/l; 5ml/l trace elements: sodium borate, 6.2mg/l; MnSO ₄ ·H ₂ O, 22.3mg/l l;ZnSO ₄ ,8.6mg/l;Na ₂ MoO ₄ 2H ₂ O,0.25mg/l;CuSO ₄ 5H ₂ O,0.025mg/l;CoCl ₂ 6H ₂ O,0.025mg/l) Cultivated umbrella grass (Cypcrus alternifolius L.) for 10 days was transferred to 400ml fresh Hoagland nutrient solution, and sodium citrate was added exogenously as a carbon source to adjust the carbon-nitrogen ratio to 7. The LR cells grown to the logarithmic phase were selected, centrifuged and washed three times with distilled water, then resuspended in deionized water, the cell concentration was adjusted to 10 ⁶ cFu/ml, and inoculated into Hoagland nutrient solution at a volume ratio of 1/100. Four treatment groups were set up in the experiment: Bacteria LR, Helicophyllum (without adding LR bacterial liquid), LR+Heliconia root exudates, LR+Helicophyllum. Umbrella grass treatment group and LR+ Umbrella grass treatment group each group adds three Umbrella grasses with the same growth, LR+ Umbrella grass root exudate treatment group adds Hoagland nutrient solution 10% volume (i.e. 40ml) Umbrella grass root exudation; Group set five replicates. On the 1st, 2nd, 3rd, and 7th day after treatment, the residual nitrogen content in the water body was determined by alkaline potassium persulfate digestion ultraviolet spectrophotometry. And on the 7th day, 1ml solution was taken from each treatment group and added to the liquid DM medium containing 50mg/l ampicillin and 50mg/l erythromycin and cultured at 28°C for 2 days. Aspirate 0.5ml of the culture solution and spread it on the BTB plate containing 50mg/l chloramphenicol. The blue colony grown on the plate was picked and shaken to extract the genomic DNA, and the 16s rDNA sequence was amplified and sent to the sequencing company for sequencing. The results showed that after 7 days, the nitrogen removal rate of the LR+ umbrella grass treatment group was significantly higher than that of the LR treatment group (Figure 7), reaching 96%. Strains LR could be isolated after 7 days in all treatment groups (except the Umberella treatment group without LR added) (Table 3).

Table 3 Survival of LR in different time periods in Hoagland nutrient solution and sewage

In the above table, "ND" means not detected, "+" means strain LR can be isolated, and "-" means strain LR is not isolated.

Example 4. Using external sewage as N eutrophication liquid to implement plant-microbe joint restoration

Sewage is taken from a water body eutrophication river course in Nanjing City, Jiangsu Province. The nitrogen content of sewage is 16.3mg-N/l. The detection method is the same as the detection method in Example 3 and is all digested with alkaline potassium persulfate by ultraviolet spectrophotometry. Determination (refer to the national standard GB11894-89). The selection of umbrella grass and thalline, the collection method of umbrella grass root exudates, the setting of treatment groups, the determination of nitrogen content in the water body are the same as in Example 3, and the volume of the test water body is 400ml. 1, 2, 3, and 7 days after the treatment, the bacteria were isolated, and the isolation steps were the same as in Example 4. The results showed that the nitrogen removal rate of the LR+Symphaea treatment group was significantly higher than that of the other three groups, and the nitrogen removal rate of the LR treatment group alone was 7%, and there was no significant difference in several time periods. The nitrogen removal rate on the second day was 45%, and there was no significant difference between the nitrogen removal rate after two days and the second day (Figure 8). The results of bacterial cell isolation showed that strain LR could be isolated from the initial treatment group of LR+Symphaea, and no strain LR could be isolated in the four time periods after the addition of strain LR alone. LR could be isolated on day 2, but could not be isolated on day 3 and day 7 (Table 3). Based on this, it is preliminarily speculated that the promotion of LR by Umbrella sp. is root exudates.

Example 5. Using external sewage as an exogenous source of N eutrophication liquid to add umbrella grass root exudates to nitrogen removal experiments

The sewage used in Example 4 was adopted, and the volume of the water body was 400ml. The selection of umbrella grass and bacteria, the collection method of umbrella grass root exudates, the determination of nitrogen content in the water body are the same as in Example 4, and three treatment groups are set: the bacteria LR treatment group, the bacteria LR+ umbrella grass root exudates treatment group , thalline LR + umbrella grass root exudates (20ml/2 days) treatment group. The bacterium LR treatment group only added bacterium LR to the sewage: select the bacterium LR grown to the logarithmic phase, wash with distilled water for three times and then resuspend in deionized water, adjust the concentration of the bacteria to 10 ⁶ cFu/ml, Inoculate into 400ml of sewage according to the volume ratio 1/100; in addition to adding the same concentration of bacteria LR as the bacteria LR treatment group, 20ml of umbrella grass rhizosphere exudates were also added to the bacterial cell LR+coryphanus root exudates treatment group; In addition to adding the same concentration of bacteria LR as the bacterial cell LR treatment group, the treatment group of cell LR + umbrella grass root exudate (20ml/2 days), after adding 20ml of umbrella grass secretion for the first time, add umbrella grass rhizosphere once every two days For secretions, the first two groups were replaced with the same volume of deionized water. The experimental results show that the nitrogen removal rate of the bacterial cell LR + umbrella grass root exudate (20ml/2 days) treatment group is the highest after 7 days, and the nitrogen removal rate of the bacterial cell LR treatment group is only 7%, and the bacterial cell LR + umbrella grass root secretion There was no significant difference in the denitrification rate of the treatment group on the 1st and 2nd day and the treatment group of LR + umbrella grass root exudates (20ml/2 days), but the denitrification rate of the 3rd and 7th day was the same as that of the 3rd day and the 7th day There was no significant difference between the two days, indicating that the denitrification of the bacteria had stopped after two days (Figure 9). Judging from the results of bacterial cell isolation: the treatment group LR + umbrella grass root exudates (20ml/2 days) can isolate the bacterial cell LR at 1, 2, 3, and 7 days after treatment, and the LR of the treatment group at four time points LR could not be isolated at any place, and LR could be isolated from the water body on the 1st and 2nd days after the treatment, but LR could not be isolated from the bacterium LR on the 3rd day and the 7th day ( Table 4).

Table 4 Survival of LR in different time periods in sewage

In the above table, "+" means that strain LR can be isolated, and "-" means that strain LR cannot be isolated.

The "bacterial strain LR" involved in the present invention all refers to the Pseudomonas LR whose preservation number is CCTCC NO: M2011159.

Claims (6)

1. A strain of denitrifying bacteria LR, classified as Pseudomonas LR (Pseudomonas sp. LR), was preserved in the China Center for Type Culture Collection on May 3, 2011, and the strain preservation number is CCTCC NO: M2011159. 2. the preservation number described in claim 1 is the application of the denitrifying bacteria LR of CCTCC NO: M2011159 in nitrogen eutrophication water pollution control. 3. The application of the denitrifying bacteria LR with the preservation number of CCTCC NO: M2011159 combined with Cyperus alternifolius L. according to claim 1 in the treatment of nitrogen eutrophication water pollution. 4. A method for carrying out plant-microbial joint restoration of nitrogen eutrophication water, characterized in that umbrella grass is planted in nitrogen eutrophication water, and the denitrification described in the inoculation number is CCTCC NO: M2011159 Bacteria LR: 7-15 strains of umbrella grass are planted per liter of water body, and 10 ml of denitrifying bacteria LR bacterial solution with a preservation number of CCTCC NO: M2011159 is inoculated with a concentration of 10 ⁶ cFu/ml per liter of water body. 5. A method for controlling nitrogen eutrophication water pollution, characterized in that the root exudates of umbrella grass are added to the nitrogen eutrophication water body, and the inoculation number is CCTCC NO: M2011159 denitrifying bacteria LR, per liter of water Add 50ml of umbrella grass root exudates to the body, inoculate 10ml of the denitrifying bacteria LR bacterial solution with the preservation number of CCTCC NO: M2011159 at a concentration of 10 ⁶ cFu/ml per liter of water body, add umbrella grass root exudates once every two days after inoculation, Add 40-60ml per liter of water each time; wherein, the root secretions of the umbrella grass are collected and prepared by the following method: after the root system of the umbrella grass is rinsed with deionized water, the relative humidity is 75%-80%, the temperature is 25 ℃, in a climate room with light intensity of 3000Lx-5000Lx, use deionized water to soak the plant root system for 4-7 hours, and use a rotary evaporator to concentrate to 1/10/volume; The mass-volume ratio is 1g: 10-30ml. 6. the method for nitrogen eutrophication water body to carry out plant-microorganism combined restoration according to claim 5, it is characterized in that the mass volume ratio of described umbrella grass root system and the deionized water used for soaking root system is 1g: 20ml.

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