CN107699513A - A kind of special degradation bacteria of black and odorous water and its application - Google Patents

Abstract

The invention provides a special degrading bacterial strain for black and odorous water and its application in treating black and odorous water. The bacterial strain is Bacillus megaterium with a preservation number of CGMCC No.13543. Ammonia nitrogen and/or phosphate removal in malodorous water bodies. The special degrading strain for black and odorous water provided by the invention has strong specificity for pollutants in black and odorous water, can efficiently utilize organic matter and ammonia nitrogen in black and odorous water, and can reduce COD of black and odorous water by more than 90%. Good phosphorus removal ability can greatly improve the purification efficiency of black and odorous water.

Description

A special degrading bacterium for black and odorous water and its application

technical field

The invention belongs to the field of microorganisms, and in particular relates to a special degrading bacterium for black and odorous water bodies and its application.

Background technique

Black and odorous water body is a general term for water bodies that exhibit unpleasant colors (black and blackish) and/or emit unpleasant odors (stinky or foul smell). In recent years, most urban rivers, closed and semi-closed pits and ponds in my country have excessive pollution. Excessive discharge directly leads to eutrophication of the water body, an imbalance of oxygen supply and consumption in the water body, and an increase in the concentration of ammonia nitrogen, hydrogen sulfide, iron, manganese sulfide, etc., resulting in continuous deterioration of water quality and the appearance of black and odorous phenomena. Water body organic pollution is one of the important factors that cause water black and odor. Organic substances such as sugars, amino acids, proteins, oils, and esters mainly exist in dissolved and suspended states. These substances are decomposed into small molecular substances under the action of microorganisms. This process consumes a large amount of dissolved oxygen. Inorganic pollutants mainly play the role of blackening in black and odorous water, and the main blackening components are easily oxidized FeS and MnS. Chemical oxygen demand (COD) reflects the degree of water pollution by reducing substances, including organic matter, nitrite, ferrous salt, sulfide, etc. Therefore, COD is a comprehensive indicator of the relative content of reducing substances .

Black and odorous water body treatment technologies mainly include artificial aeration, ecological engineering restoration, biological growth promotion, microbial purification, combined biological and plant purification and other technologies. Among them, the use of special microorganisms to metabolize high-concentration pollutants is the main method and development trend of domestic and foreign treatment of black and odorous water bodies. High-efficiency dedicated degrading bacteria can synthesize their own components by using organic matter, hydrogen sulfide, ammonia, etc. as hydrogen donors in heavily polluted water bodies, increasing dissolved oxygen, thereby purifying sewage. At present, the number of special microbial strains in black and odorous water bodies is limited, the degradation effect is uneven, and the related technology needs to be developed. Among related products, some high-efficiency special bacteria agents need to be imported, which increases the cost and affects the rapid development of the technical service market. Therefore, it is urgent to develop special microbial strains and bacterial agents for black and odorous water as soon as possible in combination with the specific needs of water treatment in my country, so as to serve relevant municipal departments and technical markets.

Contents of the invention

In order to obtain a new type of bacterial agent treatment system with high efficiency, the inventor has done a lot of work. Aiming at the typical characteristics of black and odorous water in my country, according to the natural distribution and metabolism of water microorganisms, the efficient water pollutant degradation is separated and screened from sludge. strain.

Therefore, the present invention provides a special degrading strain for black and odorous water bodies. The strain is Bacillus megaterium named AW0F5, which was preserved in No. 3, Courtyard No. 1, Beichen Road, Chaoyang District, Beijing on January 10, 2017. General Microbiology Center of the Preservation Management Committee, the deposit number is CGMCC No.13543. The 16S rDNA sequence of AW0F5 is shown in SEQ ID NO.1.

The screening steps of the above-mentioned bacterial strains in the present invention are: get sludge from Tianjin Jizhuangzi Sewage Treatment Plant, place the sample in a sterilized container and bring it back immediately after sample collection; get 5g of sludge under aseptic conditions and shake and expand in LB liquid medium Cultivate for 24 hours; take 5 mL of culture solution and transfer it to LB liquid medium for culture for 24 hours (repeat 3 times); take the last 1 mL of LB culture solution and dilute 10 ³ -10 ⁸ times, and spread the diluted solution on Gaoshi No. 1 solid culture medium base; culture at a constant temperature of 30°C, after colonies appear, repeat streaking and separation until pure colonies appear.

The present invention also provides a method for amplifying the above-mentioned special degrading strain for black and odorous water, which uses LB liquid culture medium; the culture temperature is 5-55°C, preferably 30-40°C; the pH is 6.8-8.0, preferably 7.2-7.8.

The present invention also provides another method for amplifying the above-mentioned black and odorous water-degrading strain, which uses Gaoshi No. 1 liquid medium; the culture temperature is 15-45°C, preferably 25-40°C; the pH is 6.8- 7.5.

The invention provides the application of a special degrading strain for black and odorous water bodies in the treatment of black and odorous water bodies.

In a specific embodiment of the present invention, the special strain for degrading black and odorous water reduces the COD of black and odorous water.

In another specific embodiment of the present invention, the degrading bacteria strain dedicated to black and odorous water degrades ammonia nitrogen and/or removes phosphate in black and odorous water.

In another specific embodiment of the present invention, the black and odorous water body comes from river courses, pit ponds, and ditches.

In another specific embodiment of the present invention, the pH of the black and odorous water body is 6.5-8.5, preferably 7.5-8.2; the temperature of the black and odorous water body is 10-40°C, preferably 23-30°C; the state of the black and odorous water body Standing, shaking or aeration, preferably shaking or aeration.

The present invention also provides a microbial bacterial agent for treating black and odorous water, which includes the above-mentioned special degrading bacteria for black and odorous water.

The special degrading strain for black and odorous water provided by the invention has strong specificity for pollutants in black and odorous water, can efficiently utilize organic matter and ammonia nitrogen in black and odorous water, and can reduce COD of black and odorous water by more than 90%. Good phosphorus removal ability can greatly improve the purification efficiency of black and odorous water.

detailed description

Embodiment 1: Isolation, screening and identification of strains

Sludge was taken from Tianjin Jizhuangzi Sewage Treatment Plant, and immediately after sample collection, it was placed in a sterilized container and brought back; under aseptic conditions, 5 g of sludge was shaken and expanded in LB liquid medium for 24 hours; 5 mL of culture medium was transferred to Cultivate in LB liquid medium for 24 hours (repeat 3 times); take 1 mL of the last LB culture solution and dilute it 10 ³ -10 ⁸ times, apply the diluted solution on LB solid medium, and culture at a constant temperature of 37°C, and streak repeatedly after bacteria appear Separate until pure colonies appear.

Another way of separation and screening: take sludge from Tianjin Jizhuangzi Sewage Treatment Plant, put the sample in a sterilized container and bring it back immediately after collection; take 5g of sludge in LB liquid medium under aseptic conditions and shake and expand the culture for 24 hours ;Take 5mL of the culture solution and transfer it to LB liquid medium for 24h (repeat 3 times); take the last 1mL of the LB culture solution and dilute it by 10 ³ -10 ⁸ times, and spread the dilution on Gaoshi No. 1 solid medium ; Cultivate at a constant temperature of 30°C. After colonies appear, repeat streaking and separation until pure colonies appear. .

Pick 10 pure single colonies and transfer them to LB slant medium for storage at 4°C.

Inoculate 10 strains on ammonia nitrogen enriched LB plate medium (glucose 5g, (NH ₄ ) ₂ SO ₄ 2g, NaCl 2g, FeSO ₄ 7H ₂ O 0.4g, K ₂ HPO ₄ 1g, MgSO ₄ 7H ₂ O 0.5 g, pH 7.2), cultured statically at 37°C for 3 days, in which AW0F5 survived.

Inoculate 10 strains on phosphorus-enriched LB plate medium (per 1000mL water, glucose 10g, (NH4) ₂ SO ₄ 0.5g, MgSO ₄ H ₂ O 0.3g, NaCl 0.3g, KCl 0.3g, FeSO ₄ ·7H2O 0.03g, MnSO ₄ ·H ₂ O 0.03g, K ₂ HPO ₄ 0.5g, pH 7.2), cultured statically at 37°C for 5 days, in which AW0F5 survived and appeared phosphorus-dissolving circles.

The AW0F5 strain was transferred to LB slant medium for storage at 4°C.

Gram staining of strain AW0F5 is positive. When LB medium is used, the conditions for the survival and growth of the strain are temperature 5-55° C. and pH 6.5-8.0. When cultured on a plate, the colony is round, with a smooth surface and slightly convex, regular edges, opaque milky white color, and a single bacterium is rod-shaped. After cultured in LB medium for 24 hours, the cell length was 3.0-4.0 μm. Using 16S universal primers 27F and 1492R to carry out PCR amplification of the conserved sequence of 16S bacteria, the rDNA sequence was obtained for BLAST homology comparison, and it was found that there were 97% homology with at least 15 strains of Bacillus megaterium (provided by Beijing Liuhe Huada Gene Technology Co., Ltd. Co., Ltd. sequencing), can be initially identified as Bacillus megaterium (Bacillus megaterium). The 16S rDNA sequence of AW0F5 is shown in SEQ ID NO.1.

SEQ ID NO.1:

Embodiment 2: ammonia nitrogen degradation

Preparation of ammonia nitrogen liquid medium: glucose 1g, (NH ₄ ) ₂ SO ₄ 0.4g, NaCl 2g, FeSO ₄ 7H ₂ O 0.4g, K ₂ HPO ₄ 1g, MgSO ₄ 7H ₂ O 0.5g, 1000mL deionized water, pH 7.2, sterilized at 121°C for 20min.

The AW0F5 strain was transferred from the slant medium to the LB liquid medium, and cultured at 110 r/min at 30°C for 24 hours to prepare a seed solution. The cells were collected by centrifugation at 2000r/min for 7min. Use 3mL of sterile water to prepare the bacteria suspension, take 0.5mL and inoculate it into the ammonia nitrogen liquid medium, set up a blank group without inoculation of the bacteria, and cultivate it at 23°C and 110r/min. The concentration of ammonia nitrogen was measured every 24 hours, and the degradation rate of ammonia nitrogen was calculated.

(NH ₄ ) ₂ SO ₄ (also referred to as ammonia nitrogen in this application) is the only nitrogen source in the ammonia nitrogen liquid medium, and its initial concentration of ammonia nitrogen is 85.5 mg/L. After 1 day, 7 days and 10 days, the substrate The degradation rate of ammonia nitrogen reached 55.2%, 63.9% and 85.6%.

Ammonia nitrogen degradation rate calculation formula:

Among them, X is the degradation rate of ammonia nitrogen; M1 is the concentration of ammonia nitrogen in the experimental group (mg/L); M2 is the concentration of ammonia nitrogen in the blank group (mg/L); M is the initial concentration of ammonia nitrogen (mg/L).

Example 3: Phosphorus removal

Preparation of ammonia nitrogen liquid medium: CH ₃ COONa 1g, MgSO ₄ 7H ₂ O 0.082g, FeSO ₄ 7H ₂ O 0.0037g, CaCl ₂ 0.06g, (NH ₄ ) ₂ SO ₄ 0.2g, beef extract 0.22g, K ₂ HPO ₄ 0.057g, pH7.2, sterilized at 121°C for 20min.

The strain AW0F5 was transferred from the slant medium to the LB liquid medium, and cultivated at 110 r/min at 30°C for 24 hours to prepare the seed solution. The cells were collected by centrifugation at 2000r/min for 7min. Use 3mL of sterile water to prepare the bacteria suspension, take 0.5mL and inoculate it into the ammonia nitrogen liquid medium, set up a blank group without inoculation of the bacteria, and cultivate it at 23°C and 110r/min. The concentration of phosphate was measured every 24 hours, and the phosphorus removal rate was calculated.

The initial phosphate concentration was determined to be 23.75mg/L, and the phosphorus removal rate was 57.9% 7 days after inoculation.

Phosphorus removal rate calculation formula:

Among them, Y is the phosphorus removal rate; N1 is the phosphate concentration of the experimental group (mg/L); N2 is the phosphate concentration of the blank group (mg/L); N is the initial phosphate concentration (mg/L).

Example 4: Degradation of black and odorous water raw water

The black and odorous water used in this embodiment is the water body of a pond in Qingguang Town, Beichen District, Tianjin; after measurement, the COD _Cr of the black and odorous water raw water is 240mg/L; the ammonia nitrogen content is 50mg/L; the phosphorus content is 13mg/L .

The strain AW0F5 was transferred from the slant medium to the LB liquid medium, and cultivated at 110 r/min at 30°C for 24 hours to prepare the seed solution. Bacteria were collected by centrifugation.

Use 5mL of sterile water to prepare the bacterial suspension, take 1mL and add it to the raw water of the black and odorous water body, and place it at 23°C and 50r/min. The COD, ammonia nitrogen and total phosphorus concentrations of the black and odorous raw water were measured every 24 hours, and the COD reduction rate, ammonia nitrogen degradation rate and phosphorus removal rate were calculated. The results are shown in Table 1.

Table 1 The content of each index in the water body after processing for different days

COD _Cr /mg/L Ammonia nitrogen content/mg/L Phosphorus content/mg/L 6 hours 231.42 49.35 12.28 12 hours 220.65 48.89 11.65 1 day 196.81 47.05 10.58 3 days 76.32 39.22 6.21 7 days 23.52 24.93 5.10 15 days 19.68 2.25 5.50

COD _Cr refers to the oxygen demand measured with potassium dichromate as the oxidant, and is the value of COD measured by the potassium dichromate method.

As can be seen from Table 1, the COD decreased by 68.2%, 90.2% and 91.8% respectively at 3 days, 7 days and 15 days after inoculation; The phosphorus removal rate reached 60.8% in 7 days.

A special degrading bacterium for black and odorous water provided by the present invention and its application have been introduced in detail above. In this paper, specific embodiments are used to illustrate the principle and implementation of the present invention, and the descriptions of the above embodiments are only used to help understand the method and the central idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall under the protection of the claims of the present invention.

Claims (9)

1. a kind of special degradation bacteria strains of black and odorous water, it is characterised in that the bacterial strain is bacillus megaterium, deposit number CGMCC No.13543。

A kind of 2. method for expanding the special degradation bacteria strains of black and odorous water described in claim 1, it is characterised in that use LB liquid Culture medium；Cultivation temperature is 5-55 DEG C, preferably 30-40 DEG C；PH is 6.8-8.0, preferably 7.2-7.8.

A kind of 3. method for expanding the special degradation bacteria strains of black and odorous water described in claim 1, it is characterised in that use Gao Shi 1 Number fluid nutrient medium；Cultivation temperature is 15-45 DEG C, preferably 25-40 DEG C；PH is 6.8-7.5.

4. application of the special degradation bacteria strains of black and odorous water according to claim 1 in black and odorous water is administered.

5. application according to claim 2, it is characterised in that the special degradation bacteria strains of black and odorous water reduce black and odorous water COD.

6. application according to claim 2, it is characterised in that the special degradation bacteria strains degraded black and odorous water of black and odorous water In ammonia nitrogen and/or remove phosphate.

7. application according to claim 2, it is characterised in that the black and odorous water comes from river course, swag or irrigation canals and ditches.

8. according to the application any one of claim 4-7, it is characterised in that the pH of black and odorous water is 6.5-8.5, preferably For 7.5-8.2；The temperature of black and odorous water is 10-40 DEG C, preferably 23-30 DEG C；The state of black and odorous water is standing, shakes or expose Gas, preferably shake or be aerated.

9. a kind of microbial bacterial agent for administering black and odorous water, it is characterised in that special comprising the black and odorous water described in claim 1 Degradation bacteria.