CN104862257B - A kind of method of indigenous nitrogen microbial enrichment culture and its application in ammonia nitrogen pollution in water body improvement - Google Patents

Abstract

The invention discloses a method for enriching and cultivating indigenous nitrogen-transforming microorganisms and its application in the treatment of ammonia nitrogen pollution in water bodies, belonging to the fields of environmental microbiology and environmental protection. Collect environmental samples from water bodies that need to be treated, and culture them with specific enrichment media to obtain enriched culture seeds of indigenous nitrogen-transforming microorganisms; add enriched culture seeds and enriched media to in-situ water samples for expansion Cultivate, and then apply the enriched culture seeds of the expanded culture to the water body to control the ammonia nitrogen pollution of the water body. The invention obtains high-efficiency native nitrogen-transformation microorganisms by enriching and amplifying in-situ samples, which is more targeted than general commercial bacterial agents, and also avoids the adaptability of foreign bacterial agents to the water body and potential ecological safety hazards. The present invention conducts enrichment under artificially controllable conditions to ensure the rapid growth of highly efficient native nitrogen-transforming microorganisms, which is more stable than directly adding growth-promoting agents to water bodies, and has no risk of secondary pollution.

Description

A method for the enrichment and cultivation of indigenous nitrogen-transformation microorganisms and its application in ammonia-nitrogen pollution in water bodies Applications in Governance

technical field

The invention belongs to the fields of environmental microbiology and environmental protection, and relates to a method for enriching and cultivating indigenous nitrogen-transforming microorganisms and its application in the treatment of ammonia-nitrogen pollution in water bodies.

Background technique

Industrial and agricultural production and life discharge a large amount of nutrients (such as nitrogen and phosphorus) to water bodies, resulting in eutrophication of water bodies. This is one of the most serious problems facing my country's water environment.

Ammonia nitrogen is an important indicator for monitoring and controlling eutrophication. At present, the most stringent standard for urban sewage treatment plants in my country - the first-level A discharge standard, the discharge requirement for ammonia nitrogen is 5mg/L, while the standard for ammonia nitrogen in surface water IV in my country is 1.5mg/L. This means that without follow-up advanced treatment measures, if the wastewater treated by the sewage treatment plant is discharged directly into the environment, the ammonia nitrogen in the water body will still exceed the standard. The restoration of ammonia nitrogen pollution in natural water bodies is mainly through bioremediation. Microbes are the first to be promoted in bioremediation: the main way for nitrogen to leave the water environment is through the nitrification and denitrification pathway of microorganisms, and the most critical rate-limiting step is the nitrification process of ammonia nitrogen. There are two types of microbial remediation technologies commonly used in my country. One is to put commercial microbial agents directly into polluted river water bodies. The applied microbial preparations mainly include Clear-Flo series bacterial agents from the United States, LLMO biological active liquid, effective microbial flora from Japan, photosynthetic bacteria and nitrifying bacteria from China, and have achieved certain treatment effects. The Shanghai Municipal Water Affairs Department adopted a combination of bottom aeration and dosing of microorganisms to control the Xishuangjing River, achieving the purpose of removing black odor and reducing ammonia nitrogen. Shanghai Yulei Environmental Biotechnology Co., Ltd. used "Dongjiang Actinomycetes" to restore the bottom mud of Suzhou Creek, and it also had a significant treatment effect on the eutrophic water body of Zaohe River. Although there are successful cases in the application of these bacterial agents, there are also problems that are difficult to overcome: these microbial agents are all foreign microorganisms in the environment, and there are problems of acclimatization to the soil and water, and their use effects are not very stable; even these microorganisms are also biologically invasive. Ecological security risk.

In order to overcome the shortage of microbial agents, researchers have developed another type of technology: adding microbial growth promoters (nutrients) to polluted river water to promote the growth of "indigenous" microorganisms and the metabolism of pollutants to achieve water purification the goal of. Probidic Biotechnology (Shanghai) Co., Ltd., the Department of Environmental Science and Technology of East China Normal University and Shanghai Xuhui District Environmental Protection Bureau conducted a test in a section of the river in Shangaotang, Xuhui District, using the Bioenergizer water purification and growth promoter from Probidic Solution Company of the United States. The results showed that the addition of growth promoters had an obvious and rapid effect on eliminating black and odorous water bodies and increasing dissolved oxygen (DO) in water bodies, and the nitrification rate was also significantly increased. Compared with the microbial agent technology, this technology has greatly reduced the risk of ecological safety, but it also has shortcomings: microbial growth promoters are likely to cause secondary pollution to the environment, and its growth-promoting effect is demanding on environmental conditions, so it is difficult to promote on a large scale. Therefore, its application is limited.

Contents of the invention

The purpose of the present invention is to overcome the defects and deficiencies of the prior art, provide a method for the enrichment and cultivation of indigenous nitrogen-transformation microorganisms and the application of the method in the treatment of ammonia nitrogen pollution in water bodies. The invention utilizes the enrichment medium to enrich the environmental samples of rivers or lakes that need to be treated indoors to obtain high-efficiency native nitrogen conversion microbial enrichment culture seeds, and then obtains applicable cultures through amplified culture to accelerate water body nitrogen. Transformation process to control ammonia nitrogen pollution in water bodies.

The object of the present invention is achieved through the following technical solutions:

A method for enriching and cultivating indigenous nitrogen-transforming microorganisms, comprising the following steps: collecting surface sediment from a water body to be treated, transferring it to an enrichment medium, and transferring the culture to a fresh In the enrichment medium, the shaking culture was continued; after several consecutive transfer cultures, the seeds of the enriched culture of indigenous nitrogen-transforming microorganisms were obtained. The formula of described enrichment medium is as follows:

Nutrients Dosage (per liter) (NH ₄ ) ₂ SO ₄ 0.035-0.06g K ₂ HPO ₄ 0.16-0.32g MgSO ₄ 7H ₂ O 0.0225-0.0492g NaCl 0.275-0.35g CaCl ₂ 2H ₂ O 0.06-0.083g trace elements 0.5-2mL

The pH of the above-mentioned enrichment medium is adjusted with 5% Na ₂ CO ₃ solution, so that the pH value is 7.3-7.8; wherein, the composition of trace elements is (g/L):

FeCl ₂ 4H ₂ O 2.75 CuSO ₄ 5H ₂ O 0.045 ZnSO ₄ 7H ₂ O 0.3 CoCl ₂ 6H ₂ O 0.35 MnCl ₂ 2H ₂ O 0.3 EDTA 0.5 NaMoO ₄ 2H ₂ O 0.22 NaSeO ₄ 10H ₂ O 0.01 NiCl ₂ 6H ₂ O 0.1 H ₃ BO ₄ 0.014 Na ₃ C ₆ H ₅ O ₇ 0.64 Vitamin C 0.15

The ratio of the surface sediment transferred to the enrichment medium is preferably 20-50g/200-300mL.

The shaking culture condition is preferably: 100-160 rpm shaking culture at room temperature for 2-3 days.

The ratio of the culture to the fresh enrichment medium is preferably 1:5-100 (volume ratio).

The number of culture transfers is preferably 3-5 times.

A method for expanding the culture of indigenous nitrogen-transforming microbial enrichment, comprising the following steps: collecting 30-50L of the water body overlying the source of the surface sediment in the above-mentioned method, adding 0.1-1L of the above-mentioned enrichment medium, and inoculating the above-mentioned enrichment culture Seeds 100-400mL, aerated culture. For aeration, a small air compressor is used to inject air from the bottom of the barrel through air stones, and the dissolved oxygen level is controlled at 2-4mg/L. Cultivation can use professional fermentation tanks, or ordinary plastic buckets and cement pools, but a single culture system is controlled within the range of 30-60L, and multiple culture systems can be used for mass cultivation in large water bodies.

The condition of the aerated culture is preferably aerated at room temperature for 2-5 days.

The application of the above-mentioned method for enriching and cultivating indigenous nitrogen-transforming microorganisms or the method for expanding the enrichment of indigenous nitrogen-transforming microorganisms in the treatment of ammonia nitrogen pollution in water bodies.

A method for treating water bodies polluted by ammonia nitrogen, comprising the following steps: Sprinkling the enriched culture seeds expanded and cultivated by the above method into the water bodies to be treated at a volume ratio of 1:10000-100000. During the treatment process, the dissolved oxygen level in the water should be kept above 2mg/L. When the dissolved oxygen is insufficient, artificial aeration is required.

The time for splashing the enriched culture seeds of the expanded culture is preferably in the morning when the weather is fine.

The main advantage of the present invention with respect to prior art is:

1. The present invention obtains high-efficiency native nitrogen-transformation microorganisms by enriching and amplifying in-situ samples, which is more targeted than general commercial bacterial agents, and also avoids the adaptability of foreign bacterial agents to the water body and hidden dangers to ecological safety;

2. The present invention carries out enrichment through artificially controllable conditions, which ensures the rapid growth of high-efficiency native nitrogen-transforming microorganisms, which is more stable than directly adding growth-promoting agents to water bodies, and there is no risk of secondary pollution. spectrum.

Description of drawings

Fig. 1 is the detection diagram of the enrichment and expansion culture effect of indigenous nitrogen-transforming microorganisms in the Xunsi River. The unit of the ordinate in the figure is ×10 ⁴ copy/mL.

Figure 2 is a graph showing the changes in the concentration of ammonia nitrogen in the water samples of the Xunsi River enclosure experiment.

Figure 3 is a diagram showing the results of changes in the concentration of nitrite nitrogen in the water samples of the Xunsi River enclosure experiment.

Figure 4 is a diagram showing the results of changes in the concentration of nitrate nitrogen in the water samples of the Xunsi River enclosure experiment.

Figure 5 is a diagram of the enrichment and expansion of the cultivation effect of the indigenous nitrogen-transforming microorganisms in Nanhu Lake. The unit of the ordinate in the figure is ×10 ⁵ copy/mL.

Figure 6 is a graph showing the changes in the concentration of ammonia nitrogen in the water samples of the Nanhu enclosure experiment.

Figure 7 is a graph showing the results of nitrite nitrogen concentration changes in the water samples of the Nanhu enclosure experiment.

Figure 8 is a graph showing the change of nitrate nitrogen concentration in the water samples of the Nanhu River enclosure experiment.

detailed description

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1 Improvement of water quality in Xunsi River Wai, Wuhan

Collect 100g of surface sediment samples from the Xunsi River in Wuhan City (longitude: 114°18'0", latitude: 30°29'21") with a Peterson mud extractor, put them into a ziplock bag, and quickly transport them back to the room for nitrogen Transformation microorganism enrichment culture.

The enrichment medium formula used is: (NH ₄ ) ₂ SO ₄ 0.035g, K ₂ HPO ₄ 0.16g, MgSO ₄ • 7H ₂ O 0.0225g, NaCl 0.275g, CaCl ₂ • 2H ₂ O 0.083g, trace elements 0.5 mL, distilled water 1L, 5% Na ₂ CO ₃ solution to adjust the pH so that the pH value is 7.3. Among them, the trace element formula is: FeCl ₂ • 4H ₂ O 2.75g, CuSO ₄ • 5H ₂ O 0.045g, ZnSO ₄ • 7H ₂ O 0.3g, CoCl ₂ • 6H ₂ O 0.35g, MnCl ₂ • 2H ₂ O 0.3 g, EDTA 0.5g, NaMoO ₄ • 2H ₂ O 0.22g, NaSeO ₄ • 10H ₂ O 0.01g, NiCl ₂ • 6H ₂ O 0.1g, H ₃ BO ₄ 0.014g, Na ₃ C ₆ H ₅ O ₇ 0.64g , Vitamin C 0.15g, distilled water 1L.

Inoculate 50g of freshly collected bottom sludge into 250mL enrichment medium, shake at room temperature at a shaking speed of 120rpm, and transfer 50mL of the culture to 250mL fresh enrichment medium after 3 days to continue culturing for 3 days before proceeding to the next round Enrichment transfer. After three rounds of enrichment and transfer culture, the seeds of the enriched culture of indigenous nitrogen-transforming microorganisms were obtained. Ammonia oxidizing bacteria play an important role in the process of ammonia nitrogen nitrification, so the change in the number of ammonia oxidizing bacteria was detected by fluorescent quantitative PCR during the enrichment process to indicate the enrichment and culture effect of indigenous nitrogen transforming microorganisms.

The primers used in fluorescent quantitative PCR were primers for the amoA gene of ammonia oxidizing bacteria (amoA-1F: 5'-GGGGTTTCTACTGGTGGT-3', amoA-2R: 5'-CCCCTCKGSAAAGCCTTCTTC-3'), and the amoA gene of ammonia oxidizing bacteria was cloned ( The cloning primers are the same as the fluorescent quantitative PCR primers) to the pMD18-T vector to construct an artificial plasmid as a standard sample to make a quantitative standard curve. The results of fluorescent quantitative PCR are shown in Figure 1, which indicated that the enrichment effect was very obvious. After three rounds of enrichment, the abundance of ammonia oxidizing bacteria in the culture increased from the initial 0.13×10 ⁴ copy/mL to 6.73×10 ⁹ copy/mL.

Take 30L of the overlying water sample of the Xunsi River and put it into a 50L plastic bucket, and add 0.2L of the above-mentioned enrichment medium at the same time. Inoculate 100mL of the above-mentioned enriched culture seeds into the barrel, and cultivate with aeration at room temperature. Aeration uses a small air compressor to fill air from the bottom of the barrel through air stones to maintain the dissolved oxygen in the barrel at about 3mg/L. After 3 days of aerated culture, the enriched products of the expanded culture were obtained, and the changes in the number of ammonia oxidizing bacteria were detected by fluorescent quantitative PCR to indicate the effect of the expanded culture. The results of real-time quantitative PCR are shown in Figure 1, which indicated that the expanded culture effect was very obvious, and the abundance of ammonia-oxidizing bacteria in the expanded culture product increased from 2.17×10 ⁷ copy/mL to 2.31×10 ⁹ copy/mL.

Establish four enclosures of 2m×2m in the Xunsi River, and the average water depth in the enclosures is 0.7m. Four treatment groups were set up in the experiment: group A was left standing (control group); group B was aerated; group C was inoculated with the enriched product of the expanded culture but not aerated; group D was aerated and inoculated with the enriched product of the expanded culture; The inoculum volume of group D was 1:10000 (volume ratio). The enriched product of the expanded culture is added in the morning when the weather is fine; a small air compressor is used to fill the air from the bottom of the water through the air stone for continuous aeration to maintain the dissolved oxygen in the enclosure at 2mg/L. The changes of NH ₄ ⁺ -N (ammonia nitrogen), NO ₂ ^- -N (nitrite nitrogen) and NO ₃ ^- -N (nitrate nitrogen) in the enclosure are detected every day, and the test results are shown in Figure 2-4. The results showed that under the condition of uninterrupted aeration, the conversion rate of ammonia nitrogen in the enclosures of group D was significantly higher than that of other groups; the accumulation of nitrate nitrogen and nitrite nitrogen in enclosures of group C and D was significantly higher than that of other groups; Compared with group C, although the conversion rate of ammonia nitrogen is relatively close, the accumulation of nitrate nitrogen and nitrite nitrogen is significantly lower, indicating that aeration has limited stimulation on the nitrification process, and ammonia nitrogen may be assimilated by other aerobic microorganisms.

Example 2 Experiment on enclosure of Nanhu Lake in Wuhan

Residential areas and colleges and universities around Nanhu Lake in Wuhan are densely populated, and there are many point and non-point sources of pollution; the tail water of Longwangzui sewage treatment plant is also directly discharged into Nanhu Lake; therefore, the total nitrogen and ammonia nitrogen levels in Nanhu Lake have remained high for a long time, and the water body is inferior V class water quality.

Collect 200g of surface sediment samples from Nanhu Lake (longitude: 114°21'56.83", latitude: 30°28'43.45") with a Peterson mud picker, put them into a ziplock bag, refrigerate and transport them back to the room for enrichment and cultivation of nitrogen conversion microorganisms .

The enrichment medium formula used is: (NH ₄ ) ₂ SO ₄ 0.06g, K ₂ HPO ₄ 0.32g, MgSO ₄ • 7H ₂ O 0.0492g, NaCl 0.35g, CaCl ₂ • 2H ₂ O 0.06g, trace elements 2mL , distilled water 1L, 5% Na ₂ CO ₃ solution to adjust the pH so that the pH value is 7.8. Among them, the trace element formula is: FeCl ₂ • 4H ₂ O 2.75g, CuSO ₄ • 5H ₂ O 0.045g, ZnSO ₄ • 7H ₂ O 0.3g, CoCl ₂ • 6H ₂ O 0.35g, MnCl ₂ • 2H ₂ O 0.3 g, EDTA 0.5g, NaMoO ₄ • 2H ₂ O 0.22g, NaSeO ₄ • 10H ₂ O 0.01g, NiCl ₂ • 6H ₂ O 0.1g, H ₃ BO ₄ 0.014g, Na ₃ C ₆ H ₅ O ₇ 0.64g , Vitamin C 0.15g, distilled water 1L.

Inoculate 30g of freshly collected bottom sludge into 250mL enrichment medium, shake at room temperature at a shaking speed of 120rpm, and transfer 20mL of the culture to 250mL fresh enrichment medium after 2 days to continue culturing for 2 days before proceeding to the next round Enrichment transfer. After three rounds of enrichment and transfer culture, the seeds of the enriched culture of indigenous nitrogen-transforming microorganisms were obtained. Ammonia oxidizing bacteria play an important role in the process of ammonia nitrogen nitrification, so the change in the number of ammonia oxidizing bacteria was detected by fluorescent quantitative PCR (same as Example 1) during the enrichment process to indicate the enrichment and culture effect of indigenous nitrogen-transforming microorganisms. The results of fluorescent quantitative PCR are shown in Figure 5, which indicated that the enrichment effect was very obvious. After three rounds of enrichment, the abundance of ammonia oxidizing bacteria in the culture increased from the initial 0.95×10 ⁴ copy/mL to 7.60×10 ¹⁰ copy/mL.

Take 30L of the overlying water sample of Nanhu Lake and put it into a 50L plastic bucket, and add 0.1L of the above-mentioned enrichment medium at the same time. Inoculate 200mL of enriched culture seeds into the barrel, and cultivate with aeration at room temperature. Aeration uses a small air compressor to fill air from the bottom of the barrel through air stones to maintain about 4mg/L of dissolved oxygen in the barrel. After aerated culture for 3 days, the enriched product of expanded culture was obtained. The changes in the number of ammonia oxidizing bacteria were detected by fluorescent quantitative PCR to indicate the effect of expanded culture. The results of fluorescent quantitative PCR are shown in Figure 5, which indicated that the effect of the expanded culture was very obvious, and the abundance of ammonia-oxidizing bacteria in the expanded culture product increased from 4.67×10 ⁸ copy/mL to 4.43×10 ¹⁰ copy/mL.

In this experiment, four 3m×3m enclosures were established in Nanhu Lake, and the average water depth of the enclosures was 1.6m. Four treatment groups were set up in the experiment: group A was left standing (control group); group B was aerated; group C was inoculated with the enriched product of the expanded culture but not aerated; group D was aerated and inoculated with the enriched product of the expanded culture; The amount of inoculation in group D was 1:100000 (volume ratio). The enriched product of the expanded culture should be added in the morning when the weather is fine; a small air compressor is used to fill the air from the bottom of the water through the air stone for continuous aeration to maintain the dissolved oxygen in the enclosure at 4mg/L. The changes of NH ₄ ⁺ -N, NO ₂ ^- -N and NO ₃ ^- -N in the enclosure were detected every day, and the test results are shown in Figure 6-8. The results showed that under the condition of uninterrupted aeration, the conversion rate of ammonia nitrogen and the accumulation of nitrite nitrogen in the enclosures of group C and D were relatively close, but both of them were significantly higher than those of enclosures of groups A and B; Nitrogen accumulation was significantly higher than that of the control group, but the enclosure of group D was the highest. This indicated that the addition of enrichment culture products effectively promoted the nitrification process of ammonia nitrogen in the enclosure, and the addition of enrichment culture products combined with aeration could more effectively improve the transformation efficiency.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Hubei University of Technology

<120> A method for the enrichment and cultivation of indigenous nitrogen-transforming microorganisms and its application in the treatment of ammonia-nitrogen pollution in water bodies

<130> 1

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 18

<212>DNA

<213> Artificial Sequence

<220>

<223> amoA-1F

<400> 1

ggggtttcta ctggtggt 18

<210> 2

<211> 21

<212>DNA

<213> Artificial Sequence

<220>

<223> amoA-2R

<400> 2

cccctckgsa aagccttctt c 21

Claims (10)

1. A kind of 1. method of indigenous nitrogen microbial enrichment culture, it is characterised in that comprise the following steps：Administered from needs Sediments are gathered in water body, are transferred in enriched medium, after shaken cultivation, culture is transferred to fresh enrichment In culture medium, continue shaken cultivation；Indigenous nitrogen microbial enrichment culture species are obtained after continuous several times switching culture Son；

   The formula of described enriched medium is：(NH₄)₂SO₄0.035-0.06g, K₂HPO₄0.16-0.32g, MgSO₄•7H₂O 0.0225-0.0492g, NaCl 0.275-0.35g, CaCl₂•2H₂O 0.06-0.083g, micro- 0.5-2mL, distilled water 1L, pH 7.3-7.8；Wherein, trace element formula is：FeCl₂•4H₂O 2.75g, CuSO₄•5H₂O 0.045g, ZnSO₄•7H₂O 0.3g, CoCl₂•6H₂O 0.35g, MnCl₂•2H₂O 0.3g, EDTA 0.5g, NaMoO₄•2H₂O 0.22g, NaSeO₄•10H₂O 0.01g, NiCl₂•6H₂O 0.1g, H₃BO₄0.014g, Na₃C₆H₅O₇0.64g, Vitamin C 0.15g, distilled water 1L.
2. 2. the method for indigenous nitrogen microbial enrichment culture according to claim 1, it is characterised in that：Described top layer The ratio that bed mud is transferred to enriched medium is 20-50g/200-300mL.
3. 3. the method for indigenous nitrogen microbial enrichment culture according to claim 1, it is characterised in that：Described vibration The condition of culture is：100-160rpm shaken cultivations 2-3 days at room temperature.
4. 4. the method for indigenous nitrogen microbial enrichment culture according to claim 1, it is characterised in that：Described culture The volume ratio that thing is transferred to fresh enriched medium is 1:5-100；Culture switching number is 3-5 times.
5. 5. a kind of method that indigenous nitrogen microbial enriched substance expands culture, it is characterised in that comprise the following steps：Collection power Profit requires the sediments source place water body overlying water 30-50L in 1 methods described, adds side described in 0.1-1L claims 1 Enriched medium in method, the indigenous nitrogen microbial enrichment that inoculation is obtained using any one of claim 1-4 methods described Culture seed 100-400mL, aeration culture.
6. 6. the method that indigenous nitrogen microbial enriched substance according to claim 5 expands culture, it is characterised in that：Expand Cultivating system is 30-60L.
7. 7. the method that indigenous nitrogen microbial enriched substance according to claim 5 expands culture, it is characterised in that：It is described Aeration culture condition at room temperature aeration culture 2-5 days, water body dissolved oxygen level is 2-4mg/L.
8. 8. application of the method in ammonia nitrogen pollution in water body improvement described in claim any one of 1-7.
9. A kind of 9. method for administering ammonia and nitrogen pollution water body, it is characterised in that comprise the following steps：Will be any by claim 5-7 Method described in expands enrichment culture species by volume 1 of culture:10000-100000 splashes to the water for needing to administer In body；More than water body dissolved oxygen level 2mg/L is kept in governance process.
10. 10. the method according to claim 9 for administering ammonia and nitrogen pollution water body, it is characterised in that：In the morning of fair weather,

    Enrichment culture species that will be enlarged by culture is splashed into the water body for needing to administer.