CN113528382B

CN113528382B - Construction and application of composite denitrification microbial inoculum capable of utilizing low-cost carbon source

Info

Publication number: CN113528382B
Application number: CN202110757936.9A
Authority: CN
Inventors: 张宏耀
Original assignee: China Complete Equipment Import And Export Group Co ltd
Current assignee: China Complete Equipment Import And Export Group Co ltd
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2023-04-14
Anticipated expiration: 2041-07-05
Also published as: CN113528382A

Abstract

The invention relates to a composite denitrifying microbial inoculum, which comprises heterotrophic nitrification-aerobic denitrification bacteria D1 and aerobic acid-producing bacteria W1; wherein the heterotrophic nitrification-aerobic denitrification bacterium D1 is Alcaligenes aquatilis with a preservation number of CGMCC No.21580; the aerobic acid-producing bacterium W1 is a Microbacterium laevaniformans (Microbacterium laevaniformans) with the preservation number of CGMCC No.19762. The invention also relates to application of the composite denitrification bacterial agent in pollutant denitrification. The composite denitrifying bacterial agent successfully solves the dependence of heterotrophic nitrification-aerobic denitrifying bacteria D1 on high-cost carbon sources such as small molecular acid and the like by utilizing the functional interaction relationship of the two strains, greatly saves the cost of adding the carbon sources in the practical denitrification application, and can be applied to the denitrification of domestic sewage, wherein the ammonia nitrogen removal rate reaches 84%, and the total nitrogen removal rate reaches 48%.

Description

Construction and application of composite denitrification microbial inoculum capable of utilizing low-cost carbon source

Technical Field

The invention relates to an aerobic denitrification composite bacterial agent for breaking through carbon source utilization limitation and a preparation method and application thereof, belonging to the technical field of bioremediation of environmental pollution.

Background

Microbial denitrification mainly comprises three types: (1) Denitrogenation by a traditional autotrophic nitrification-anaerobic denitrification system; (2) anammox strain process; and (3) denitrifying by using the heterotrophic nitrification-aerobic denitrification strain.

In recent years, many microorganisms have been found to have heterotrophic nitrification-aerobic denitrification, and these microorganisms have advantages over the former two denitrification systems and strains in that (1) the reaction procedure is simple, and nitrification and denitrification can be performed simultaneously; (2) the environment adaptability is strong, and the growth speed is high; (3) As the alkalinity generated in the denitrification process can partially compensate the alkalinity consumption in the nitrification process, the buffer amount required in the denitrification process is less. Therefore, the denitrification way of the heterotrophic nitrification-aerobic denitrification bacteria has the advantages of high efficiency, low equipment requirement, small occupied space and the like, and has wider application prospect.

However, these strains often use small-molecule acids as carbon sources, and table 1 exemplifies representative heterotrophic nitrification-aerobic denitrification strains and their removal capabilities that have been published previously. The strain has excellent ammonia nitrogen and total nitrogen removal effects only in the presence of small molecular acids such as acetic acid, succinic acid and citric acid, but if glucose is used as a unique carbon source, all the report strains do not have denitrification, the stability of the strain in the practical application process is influenced by the limitation of the utilization of the carbon source, and the popularization and application of the strain are greatly hindered. In addition, from the economic perspective, the low molecular acid greatly increases the cost of strain fermentation due to higher cost, seriously affects the effect of practical application, and is not beneficial to large-scale popularization.

Table 1 reports the carbon source utilization ability and denitrification effect of the strain having heterotrophic nitrification-aerobic denitrification ability

Disclosure of Invention

The invention provides a composite microbial inoculum which can utilize glucose as a unique carbon source for denitrification and promotes the efficient utilization of heterotrophic nitrification-aerobic denitrification strain resources in the aspect of biological denitrification by rational design and screening and based on the interaction of metabolites of aerobic acid-producing bacteria W1 and heterotrophic nitrification-aerobic denitrification bacteria D1, thereby breakthrough solving the limitation of the heterotrophic nitrification-aerobic denitrification bacteria on the utilization of the carbon source. Meanwhile, in the practical application process, the traditional separate strain fermentation is changed into two strains for co-fermentation by using cheap glucose, and the cheap glucose is used for replacing expensive micromolecular acid as the carbon source, so that the application cost is greatly saved.

Specifically, the invention provides the following technical scheme:

1. the composite denitrifying microbial inoculum is characterized by comprising heterotrophic nitrification-aerobic denitrification bacteria D1 and aerobic acid-producing bacteria W1;

wherein the heterotrophic nitrification-aerobic denitrification bacterium D1 is Alcaligenes aquatilis, and the preservation number is CGMCC No.21580;

the aerobic acid-producing bacterium W1 is a levan-producing Microbacterium (Microbacterium laevanesormans) with the preservation number of CGMCC No.19762.

2. The composite denitrifier according to item 1, wherein the volume ratio of the heterotrophic nitrification-aerobic denitrification bacteria D1 to the aerobic acid producing bacteria W1 is 1:5 to 1, preferably 1 to 1.

3. The method for preparing a composite denitrifying bacterial agent according to any of items 1 to 2 is characterized in that heterotrophic nitrification-aerobic denitrification bacteria D1 and aerobic acid-producing bacteria W1 are fermented and cultured respectively, and then fermentation liquids are mixed in proportion.

4. The production method according to item 3, wherein the volume ratio of the heterotrophic nitrification-aerobic denitrification bacteria D1 to the aerobic acid producing bacteria W1 is 1:5 to 1, preferably 1 to 1.

5. The preparation method according to item 3, wherein the method further comprises adsorbing the mixed fermentation broth onto a substrate, and then drying and granulating at a low temperature; the substrate is preferably diatomaceous earth or zeolite powder.

6. The composite denitrification microbial inoculum of any one of items 1-2 is applied to pollutant denitrification.

7. A method for denitrifying pollutants, comprising adding the complex denitrification microbial inoculum according to item 1 or 2 to pollutants.

8. The method of item 7, wherein the dissolved oxygen of the contaminants is maintained at 3mg/L to 8mg/L during the denitrification.

9. The method of item 7, wherein the contaminant is sludge or sewage.

[ biological Material Collection ]

The heterotrophic nitrification-aerobic denitrification strain D1 of the invention is stored in:

china center for type culture Collection/China Committee for culture Collection of microorganisms common microbiological center (CGMCC), wherein the preservation date is 2021 year, month 1 and 18 days, and the preservation number is CGMCC No.21580; the address of the depository is: xilu No.1, beijing, chaoyang, beijing, and institute for microbiology, china academy of sciences.

The aerobic acid-producing strain W1 of the invention is preserved in:

china center for type culture Collection/China Committee for culture Collection of microorganisms common microbiological center (CGMCC), wherein the preservation date is 30 days 4 months 2020, and the preservation number is CGMCC No.19762; the address of the depository is: xilu No.1, beijing, chaoyang, beijing, and institute for microbiology, china academy of sciences.

Technical effects

1. Screening of excellent strains: the invention screens out heterotrophic nitrification-aerobic denitrification strains with high-efficiency denitrification and simultaneously screens out strains with aerobic acid production capability;

2. by means of the interaction of the strains, the limitation of carbon source utilization of heterotrophic nitrification-aerobic denitrification strains is broken through, and the stability of the synchronous nitrification-denitrification strains in practical application is improved;

3. cheap carbon sources such as glucose and the like are used for replacing relatively expensive organic micromolecular acid, so that the cost of adding the carbon sources in practical application and the cost of large-scale fermentation are greatly saved.

In a word, the composite denitrification microbial inoculum comprises two strains, namely heterotrophic nitrification-aerobic denitrification bacteria D1 and aerobic acid-producing bacteria W1, wherein the dependence of the heterotrophic nitrification-aerobic denitrification bacteria D1 on high-cost carbon sources such as small molecular acids is successfully solved by utilizing the functional interaction relationship of the two strains, and the cost of adding the carbon sources in the practical denitrification application is greatly saved by taking low-cost glucose as the denitrification carbon source. The composite denitrifying bacteria agent can be applied to denitrification of domestic sewage, and the removal of ammonia nitrogen reaches 84% and the removal of total nitrogen reaches 48%.

Drawings

FIG. 1 shows the denitrification effect of the co-fermentation culture solution obtained by the fermentation of the complex microbial inoculum of the invention on domestic sewage.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the accompanying drawings in combination with the embodiments.

Definition of

Heterotrophic nitrification-aerobic denitrification (heterotrophic nitrification and aerobic denitrification)The bacteria refer to a type of bacteria capable of heterotrophic nitrification and denitrification in the presence of oxygen, and can not only convert ammonia nitrogen into gaseous products, but also can not cause NO ₂ ^- -N/NO ₃ ^- Accumulation of N, and removal of COD from the wastewater.

The aerobic acid-producing bacteria refer to strains which grow by using a cheap carbon source (such as glucose) under aerobic conditions and simultaneously produce a large amount of small molecular acid; in the invention, the obtained metabolite can provide sufficient growth and biochemical factors for the denitrifying strains.

Example 1 Strain screening

The invention provides an aerobic denitrification compound microbial inoculum, and particularly breaks through the limitation of carbon source utilization of a heterotrophic nitrification-aerobic denitrification microbial inoculum. The invention relates to a composite microbial inoculum, which relates to two strains, namely W1 and D1, wherein W1 is aerobic acid-producing bacteria, the acetic acid can be produced by utilizing glucose under the aerobic condition, the dissolved oxygen range is 1-8 mg/L, the content of the acetic acid produced in 24h is about 0.5mM, and the sequencing comparison result of 1696 is Rhodococcus sp; d1 is heterotrophic nitrification-aerobic denitrification bacteria, can utilize small molecular acid such as acetic acid, succinic acid and the like to carry out high-efficiency synchronous nitrification and denitrification under aerobic conditions, and can grow rapidly under heterotrophic conditions, the ammonia nitrogen removal rate is as high as 100%, the total nitrogen removal rate is as high as 45%, and the 16S rDNA sequencing comparison result is Alcaligenes sp.

1. The separation and purification method and the functional verification of the heterotrophic nitrification-aerobic denitrification bacteria D1 are as follows:

enrichment medium for D1: 10mg/L NH ₄ Cl，20mg/L CH ₃ COONa，0.2mg/L MgSO ₄ ·7H ₂ O,0.2mg/L yeast extract (Diamond), 0.2mg/L peptone (Diamond), 1mg/L EDTA-Na ₂ 1.5mg/L sodium pyruvate, adjusting pH to 7.5, 121 ℃, and autoclaving for 15 min; 10mL of filter sterilized 0.1g/L NaHCO was added to the medium ₃ Solution and 20mg/L KH ₂ PO ₄ And (3) solution.

Isolation medium for D1: 0.5g/L yeast extract (Diamond), 0.5g/L peptone (Diamond), 0.5g/L casein hydrolysate (Diamond), 0.5g/L glucose, 0.5g/L soluble starch (Diamond), 0.3g/L potassium dihydrogen phosphate 0.03g/L, anhydrous magnesium sulfate, 0.3g/L sodium pyruvate, pH 7.0.

Functional validation medium for D1: 0.1g/L (NH) ₄ ) ₂ SO ₄ ，0.7g/L CH ₃ COONa，0.50g/L KH ₂ PO ₄ ,0.50g/L Na ₂ HPO ₄ ，0.20g/L MgSO ₄ ·7H ₂ 0.20g of O, 2.00mL of trace elements and pH 7.3. Wherein each 1L of the trace elements comprises: EDTA-2Na 57.10g, znSO ₄ ·7H2O 3.90g，CaCl ₂ ·2H ₂ O 7.00g,MnCl ₂ ·4H ₂ O 1.00g,FeSO ₄ ·5.00g，(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O 1.10g，MnSO ₄ ·4H ₂ O 1.60g，CoCl ₂ ·6H ₂ O 1.60g,pH 6.0。

Collecting 20g of lake water sample and sediment mixed sample, enriching in a D1 enrichment medium for half a month, taking enrichment liquid for 10 times of gradient dilution, coating in a D1 separation medium, and selecting single colonies with different forms for seed preservation. And then respectively activating the pure bacteria, inoculating the pure bacteria into a D1 functional verification culture medium according to the proportion of 1/100, measuring the denitrification capacity of the pure bacteria within 48 hours, and finally screening out a D1 strain with the preservation number of CGMCC No.21580.

2. The separation and purification method and the functional verification of the aerobic acid-producing bacteria W1 are as follows:

enrichment medium for W1: 0.5g/L KH ₂ PO ₄ ，1g/L Na ₂ HPO ₄ ，0.2g/L MgSO ₄ ·7H ₂ O,1g/L glucose, 1.07g/L NH ₄ Cl,0.03g/L yeast extract and 2mL trace elements, and adjusting the pH to 7.3; wherein, the trace elements (1L): EDTA-2Na 0.5g, znSO ₄ ·7H ₂ O 0.22g，CaCl ₂ ·2H ₂ O 0.055g，MnCl ₂ ·4H ₂ O 0.051g，FeSO ₄ ·0.0499g，(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O 0.011g，MnSO ₄ ·4H ₂ O 0.0157，CoCl ₂ ·6H ₂ O 0.016g,pH 6.0。

The isolation medium for W1 was the same as the W1 enrichment medium.

Collecting 5g of lake sediment in 100mL of W1 enrichment medium, carrying out enrichment at 30 ℃ and 150rpm, transferring according to 5 percent, and transferring for one generation in two days and 7 generations in total. After the enrichment culture is finished, the subculture sample is diluted and then spread in a solid W1 isolation medium (1.5% agar is added on the basis of the W1 isolation medium) to carry out strain isolation and purification. Then selecting a culture strain to inoculate in a W1 separation culture medium, measuring the pH value of the culture medium after 3 days, detecting the acetogenic capability through reverse high performance liquid chromatography, finally screening two acid-producing strains of W1 and W2, and finally determining and selecting the W1 strain according to a functional compatibility experiment with D1, wherein the preservation number is CGMCC No.19762.

3. The compatibility proportion of the two bacteria is as follows: selecting D1 functional verification culture medium with compatible culture medium, and adding carbon source CH ₃ COONa was replaced by glucose, and the C/N ratio was adjusted to 15, pH 7.5. D1 cannot utilize glucose and cannot grow in the culture medium, and W1 can grow in the culture medium, and the glucose is converted into small molecular acid for D1 to utilize, thereby playing a denitrification function.

The invention selects W1 and D1 to carry out proportion verification according to the proportion of 20.

4. Fermentation production:

seed fermentation medium: 5g/L tryptone, 5g/L yeast extract, 5g/L NaCl.

Co-fermentation medium: 20g/L glucose, 5g/L corn steep liquor, 1g/L urea and K ₂ HPO ₄ 1g/L，MgSO ₄ 0.5g/L，pH 7.5。

Controlling a symbiotic culture process: mixing W1 and D1 according to the proportion of 10.

5. The fermentation and application effects are as follows:

when the co-fermentation culture medium is used alone for culture, the D1 alone cannot grow, and the W1 alone does not have denitrification capability;the two are combined to carry out co-fermentation to obtain the strain OD ₆₀₀ 18, the domestic sewage is treated by utilizing the fermentation culture according to one thousandth of proportion, the ammonia nitrogen removal rate can reach 99 percent, the total nitrogen removal rate is over 50 percent, and only a small amount of nitrite and nitrate are accumulated.

Example 2: breaking through carbon source utilization limitation through microbial inoculum interaction

Firstly, the method of the embodiment 1 is adopted to screen and obtain the heterotrophic nitrification-aerobic denitrification bacteria D1 with the preservation number of CGMCC No.21580 and the aerobic acid-producing bacteria W1 with the preservation number of CGMCC No.19762, and then the heterotrophic nitrification-aerobic denitrification bacteria D1 and the aerobic acid-producing bacteria W1 are frozen and stored at the temperature of minus 80 ℃ respectively for standby.

The heterotrophic nitrification-aerobic denitrification bacteria D1 with the preservation number of CGMCC No.21580 and the aerobic acid-producing bacteria W1 with the preservation number of CGMCC No.19762 which are respectively frozen in the culture medium LB solid medium are streaked and activated, after 24 hours of growth at 30 ℃, single clones are respectively picked and cultured in the LB liquid medium for expansion, the culture medium is stirred at 30 ℃,200rpm and 24 hours.

Respectively taking 10mL of D1 and W1 culture medium liquid, centrifuging at 6000rpm for 10min, suspending with sterile phosphate buffer solution, centrifuging, adjusting OD of thallus with phosphate buffer solution ₆₀₀ Is 2. Mixing according to the proportion that D1: W1 is 1:1 and 1 is 10, then respectively connecting the mixed solution to a compatible medium shake flask according to the proportion of 1/100, repeating each experiment for 3 times, culturing at 30 ℃ and 150rpm, respectively sampling at 24h and 48h to determine the results of ammonia nitrogen and total nitrogen, and using a sample only connected with D1 and a sample only connected with W1 as a control.

The results are shown in table 2, and it can be seen from table 2 that: the independent D1 strain cannot grow on a culture medium only using glucose as a carbon source, so ammonia nitrogen and total nitrogen are unchanged; the single W1 strain grows well, but does not have denitrification capacity, and only assimilates partial ammonia nitrogen due to the growth of the strain; after the D1 and the W1 are mixed in a proportion of 1:1, ammonia nitrogen and total nitrogen are reduced compared with the independent W1, and the denitrification rate is limited, which indicates that the D1 strain in the proportion cannot play a high-efficiency denitrification function, probably because the W1 strain is small in proportion and weak in acid production capacity and is not enough to provide the growth and the function of the D1; after D1 and W1 are mixed according to a ratio of 1.

TABLE 2

Example 3: microbial inoculum co-fermentation by using glucose as cheap carbon source

Respectively carrying out amplification culture on aerobic acid-producing bacteria W1 with the preservation number of CGMCC No.19762 and heterotrophic nitrification-aerobic denitrification bacteria D1 with the preservation number of CGMCC No.21580 by using a seed fermentation culture medium, mixing the two strains according to the proportion that the ratio of W1 to D1 is 10. Meanwhile, setting a control group, wherein the control group is formed by that fermentation seeds are not mixed and are respectively inoculated in a fermentation tank according to the proportion of 1/100, wherein W1 utilizes a co-fermentation culture medium, D1 fermentation culture medium is formed by replacing a carbon source glucose with sodium acetate on the basis of the co-fermentation culture medium and is fermented according to a co-fermentation culture process to respectively obtain W1 single fermentation liquid and D1 single fermentation liquid.

After the fermentation is finished, taking the co-fermentation culture solution, the W1 single fermentation solution and the D1 single fermentation solution, respectively adjusting the OD600 to be 5, and mixing the W1 single fermentation solution and the D1 single fermentation solution according to a ratio of 10. 4 fermentation liquors of the co-fermentation culture solution, the mixed fermentation liquor, the W1 single fermentation liquor and the D1 single fermentation liquor are respectively inoculated into a functional culture medium for shaking the flask according to the proportion of 1/1000, and the denitrification rate is measured after 48 hours.

The results, as shown in table 3, show: the total nitrogen of the fermentation broth groups W1 and D1 alone did not change significantly from that before inoculation, because W1 did not provide denitrification, and D1 did not grow on the compatible media. The total nitrogen removal of the co-fermentation culture solution group and the mixed fermentation solution group respectively reaches 55 percent and 51 percent, which shows that cheap glucose replaces relatively expensive organic micromolecular acid as a carbon source to carry out co-fermentation on W1 and D1, so that the cost of practical application is saved, and the stability of aerobic denitrification is achieved.

TABLE 3

Experimental group	Ammonia nitrogen (mg/L)	Total nitrogen (mg/L)
			Co-fermentation culture solution	0	9.07±0.08
Mixed fermentation liquor	0	9.89±0.05
			W1 fermentation broth alone	5.51±0.05	21.43±1.25
D1 fermentation broth alone	19.66±0.01	20.57±1.01

Example 4: the composite microbial inoculum is used for denitrification of domestic sewage under laboratory conditions;

the method adopts the sewage of a certain Beijing sewage treatment plant with 1mm sieve pores, and the ammonia nitrogen is 44mg/L and the total nitrogen is 54mg/L through determination. 30L of sewage was taken and added to a reactor, an aeration apparatus was added to maintain the dissolved oxygen in the water at 5mg/L, and the co-fermentation broth obtained in example 3 (mixed fermentation at a W1: D1 ratio of 10: 1) was added to the reactor at a ratio of 1/1000, while the reactor without the broth was used as a control. Samples were taken every 24 hours for a total of 5 days.

As shown in FIG. 1, the ammonia nitrogen removal rate was 84% or more, the total nitrogen removal rate was about 48%, and the denitrification effect was good after 5 days.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The composite denitrifying bacterial agent is characterized by consisting of heterotrophic nitrification-aerobic denitrification bacteria D1 and aerobic acid-producing bacteria W1;

wherein the heterotrophic nitrification-aerobic denitrification bacterium D1 is Alcaligenes aquatilis with a preservation number of CGMCC No.21580;

2. The composite denitrificaion agent according to claim 1, wherein the volume ratio of the heterotrophic nitrification-aerobic denitrification bacteria D1 to the aerobic acid producing bacteria W1 is 1:5 to 1, or 1.

3. The method for preparing a composite denitrification microbial inoculum of any one of claims 1-2, wherein heterotrophic nitrification-aerobic denitrification bacteria D1 and aerobic acid-producing bacteria W1 are fermented and cultured respectively, and then the fermentation liquids are mixed in proportion.

4. The preparation method according to claim 3, wherein the volume ratio of the heterotrophic nitrification-aerobic denitrification bacteria D1 to the aerobic acid producing bacteria W1 is 1:5 to 1, or 1.

5. The method of claim 3, further comprising adsorbing the mixed fermentation broth onto a substrate, and then drying and granulating at a low temperature.

6. The method of claim 5, wherein the substrate is diatomaceous earth or zeolite powder.

7. The use of the complex denitrification bacterial agent of any one of claims 1-2 for denitrification of pollutants.

8. A method for denitrogenation of a pollutant, comprising adding the composite denitrogenation microbial agent of claim 1 or 2 to the pollutant.

9. The method of claim 8, wherein dissolved oxygen of the contaminants is maintained at 3mg/L to 8mg/L during denitrification.

10. The method of claim 8, wherein the contaminant is sludge or sewage.