CN110699350A

CN110699350A - Immobilized microbial inoculum and preparation method and application thereof

Info

Publication number: CN110699350A
Application number: CN201910718561.8A
Authority: CN
Inventors: 陈惠明; 蒋自胜; 翟德勤; 余艳鸽; 郑茂盛; 商放泽; 俞静雯
Original assignee: PowerChina Eco Environmental Group Co Ltd
Current assignee: PowerChina Eco Environmental Group Co Ltd
Priority date: 2019-08-05
Filing date: 2019-08-05
Publication date: 2020-01-17
Anticipated expiration: 2039-08-05
Also published as: CN110699350B

Abstract

The invention relates to the technical field of microbial inoculum immobilization, and particularly discloses an immobilized microbial inoculum, and a preparation method and application thereof. The immobilized microbial inoculum comprises acinetobacter and an immobilized carrier, wherein the immobilized carrier is rice bran. The preparation method of the immobilized microbial inoculum at least comprises the following steps: culturing the acinetobacter to obtain a bacterial suspension; and sterilizing the bran coat, uniformly mixing the bran coat and the bacterial suspension under the aseptic condition, and drying to obtain the immobilized microbial inoculum. According to the invention, the immobilized microbial inoculum is prepared, and the acinetobacter is loaded into the immobilized carrier, so that the physiological activity and stability of the microorganism are not affected, and an aerobic and anoxic microenvironment is provided for the microorganism, thereby being beneficial to the growth of the microbial inoculum and the performance of denitrification and denitrification of the microbial inoculum.

Description

Immobilized microbial inoculum and preparation method and application thereof

Technical Field

The invention relates to the technical field of microbial inoculum immobilization, in particular to an immobilized microbial inoculum and a preparation method and application thereof.

Background

The rapid development of industry and agriculture and the production activities of human beings bring serious pollution to the water environment. The exceeding of nitrogen is the most common pollution problem in water body pollution, which can cause eutrophication of water body, cause mass propagation of algae, reduce dissolved oxygen in the water body and cause black and odorous water body, thereby not only endangering the water safety of human beings, but also seriously influencing the ecological balance of the water body; in addition, excessive nitrogen discharge can cause the nitrogen balance to be destroyed, thereby causing the water body to lose the self-cleaning function. Therefore, the sewage rich in nitrogen can be discharged into the natural environment after the sewage is subjected to denitrification treatment and reaches the standard.

Biological denitrification is a denitrification method widely applied at present and has the advantages of good treatment effect, stable and reliable treatment process, convenient operation and management and the like. The traditional biological denitrification is completed by utilizing the aerobic autotrophic nitrification of nitrifying bacteria and the anaerobic heterotrophic denitrification of denitrifying bacteria, and the biological denitrification needs to provide two reactors with oxygen and oxygen respectively to carry out nitrification and denitrification respectively so as to achieve the aim of denitrification. Therefore, the reactor occupies a large space, the operation cost is high, and the process is complicated.

Immobilization is the physical or chemical means to confine free microorganisms or enzymes in a specific spatial region for more widespread and effective use. In the sewage with flow dynamics, because the flowing of the water body, the selection of the immobilized carrier material directly influences the physiological activity and stability of the microorganism, even the loss of the microorganism, the selection of the immobilized carrier which can not only ensure enough microorganism amount in unit volume, but also protect the microorganism to be in a high activity state becomes a hotspot of research.

Disclosure of Invention

Aiming at the problems of high cost, complex process, reduction of strain activity by an immobilization process and the like of the existing biological denitrification, the invention provides an immobilized microbial inoculum.

And a method for producing the immobilized microbial agent.

And the application of the immobilized microbial inoculum in the treatment of nitrogen-containing sewage.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

an immobilized bacteria agent comprises acinetobacter and an immobilized carrier, wherein the acinetobacter is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO. 16620; the immobilized carrier is rice bran.

The immobilized microbial inoculum provided by the invention has the following advantages:

the invention preserves the acinetobacter in the form of immobilized bacteria agent, loads the acinetobacter into the bran coat, solves the problem of difficult preservation of the acinetobacter, ensures that the physiological activity and stability of the microorganism are not affected, provides an aerobic and anoxic microenvironment for the microorganism, is beneficial to the growth of the bacteria agent and the performance of denitrification and denitrification of the bacteria agent, is also beneficial to keeping high viable bacteria concentration and improving the rate of degrading nitrogen-containing sewage when in use, and has the advantages of convenient application, separation and repeated use.

The invention takes the bran coat with small mass as the immobilized carrier, and is easily influenced by wind when being scattered to the water body, so that the immobilized microbial inoculum can be uniformly dispersed into the sewage, floats on the water surface, and is not easy to sink into the water to cause secondary pollution to the water body; meanwhile, the bran coat has obvious porous characteristic, large specific surface area and strong adsorption capacity, and after the immobilized microbial agent taking the bran coat as a carrier is added into sewage, the immobilized microbial agent can promote nitrogen-containing pollutants to migrate to the bran coat carrier, so that the bran coat can simultaneously enrich acinetobacter and the pollutants, and the contact between microorganisms and the pollutants is increased; the bran coat can also provide an independent microenvironment for the acinetobacter, so that the direct invasion of external adverse factors to the acinetobacter is shielded, and the removal efficiency of the acinetobacter to nitrogen-containing pollutants in a polluted environment is improved; and the bran coat as an organic matter can provide nutrient substances for the propagation of the acinetobacter and provide a nutritional basis for keeping high viable bacteria concentration of the acinetobacter, so that the bran coat immobilized microbial inoculum can realize the integration of enrichment and degradation of pollutants, and is beneficial to promoting the degradation of nitrogen-containing pollutants in sewage.

The Acinetobacter provided by the invention is an aerobic denitrifying bacterium, the Acinetobacter is obtained by enrichment screening through a biological culture means, the Acinetobacter has the characteristic of playing a role in denitrification under aerobic conditions, and the aerobic denitrifying bacterium strain is adopted to replace the conventional anaerobic denitrifying bacterium, so that the nitrification and the denitrification can be carried out in one reaction when the biological denitrification treatment of sewage is carried out, thereby simplifying the sewage treatment process, reducing the capital construction operation cost and improving the biological denitrification effect, and the Acinetobacter has good application prospect in the aspect of nitrogen-containing sewage treatment.

Preferably, the nucleotide sequence of 16S rDNA of Acinetobacter has a sequence as shown in SEQ ID No. 1.

The 16S rDNA gene sequence of the acinetobacter provided by the invention is shown as follows:

SEQ ID NO.1：

GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATTCTGATCCGCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGATCGGCTTTTTGAGATTAGCATCCTATCGCTAGGTAGCAACCCTTTGTACCGACCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCGTCCCCGCCTTCCTCCAGTTTGTCACTGGCAGTATCCTTAAAGTTCCCGACATTACTCGCTGGCAAATAAGGAAAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTATGTAAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTTACTATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAGTCTTGCGACCGTACTCCCCAGGCGGTCTACTTATCGCGTTAGCTGCGCCACTAAAGCCTCAAAGGCCCCAACGGCTAGTAGACATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCATGCTTTCGCACCTCAGCGTCAGTGTTAGGCCAGATGGCTGCCTTCGCCATCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCATCCTCTCCCACACTCTAGCTAACCAGTATCGAATGCAATTCCCAAGTTAAGCTCGGGGATTTCACATTTGACTTAATTAGCCGCCTACGCGCGCTTTACGCCCAGTAAATCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGCGAGTAACGTCCACTATCTCTAGGTATTAACTAAAGTAGCCTCCTCCTCGCTTAAAGTGCTTTACAACCATAAGGCCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCGGATCATCCTCTCAGACCCGCTACAGATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCCGACTTAGGCTCATCTATTAGCGCAAGGTCCGAAGATCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCATTCCTTTCGAAATGTTGTCCCCCACTAATAGGCAGATTCCTAA。

the length of the sequence is 1282bp, and the accession number of the gene sequence in GenBank is MK 163532. The gene sequence of the strain is uploaded to GenBank for comparison, and the comparison result shows that: the gene sequence of the strain has the highest similarity with the sequence of Acinetobacter. Therefore, the strain is judged to belong to Acinetobacter (Acinetobacter) by combining morphological characteristics of the bacteria, and is named as Acinetobacter sp.a 3.

Specifically, the strain Acinetobacter sp.A3 of the invention is deposited in China general microbiological culture Collection center (CGMCC) at 24/10/2018, and the address is as follows: the preservation number of the Xilu-Shi is CGMCC NO. 16620.

Preferably, the immobilized microbial agent is used for loading the thallus in the bacteria suspension of the acinetobacter onto the immobilized carrier by adopting an adsorption method.

The application adopts the adsorption method to directly utilize the acinetobacter to carry out loading, removes the complicated extraction step of the acinetobacter, and has convenient manufacture and low cost.

Preferably, the mass ratio of the immobilization carrier to the bacteria suspension of acinetobacter is 4.5-5.5: 1.5-2.5.

Preferably, the proportion of the immobilized carrier and the bacteria suspension is selected, so that the quantity of the acinetobacter in the immobilized bacteria agent is moderate, and if the quantity of the acinetobacter is too small, the degradation rate is influenced; if the number of the acinetobacter is too large, the immobilized carrier is supersaturated for adsorbing the microorganisms, the microorganisms are desorbed to enter a water body in the process of degrading pollutants, however, the physiological activity of the microorganisms in the water body is low, and the stability is poor, so that the proportion of the immobilized carrier and the bacterial suspension is preferably controlled.

Preferably, the water absorption of the bran coat is 35-45%.

Under the condition of the same mass, the carrier with higher water absorption rate can absorb more bacterial suspension; however, if the water absorption of the bran coat is too high, the microbial adsorption capacity is too high, the immobilized microbial agent can be desorbed when entering a water body, so that the waste of the acinetobacter is caused, the bran coat and the bacterial suspension are difficult to be uniformly mixed, and whether the distribution of the microbes in the bran coat is uniform or not is difficult to be ensured, so the proportion of the bran coat is optimized, and the microbial adsorption capacity of the immobilized carrier is moderate.

Preferably, the bacterial suspension is a bacterial suspension obtained by culturing the acinetobacter to a logarithmic growth phase, and the OD600 of the bacterial suspension is 0.60-0.63. And measuring the absorbance value of the bacterial suspension at the wavelength of 600nm by using an ultraviolet spectrophotometer, and recording as OD 600.

The growth speed of the microorganism in the logarithmic growth phase is the maximum, the number of cells is increased exponentially, and the selection of the bacterial suspension in the logarithmic growth phase can ensure that the prepared immobilized microbial inoculum has high growth speed and high biological activity.

And verifying the content of the acinetobacter in the bacterial suspension by using the OD600 value, and further controlling whether the bacterial suspension is in a logarithmic growth phase.

Furthermore, the invention also provides a preparation method of the immobilized microbial inoculum. The preparation method at least comprises the following steps:

step a, culturing the acinetobacter to obtain a bacterial suspension;

and b, sterilizing the bran coat, uniformly mixing the bran coat and the bacterial suspension under the aseptic condition, and drying to obtain the immobilized microbial inoculum.

Compared with the prior art, the preparation method of the immobilized microbial inoculum provided by the invention has the following advantages:

the invention loads the acinetobacter onto the bran coat by an adsorption method, has simple preparation method and mild conditions, is beneficial to industrial popularization, fixes the microorganism in the bran coat, and improves the biological activity and stability of the immobilized microbial inoculum by utilizing the characteristics of the bran coat.

Preferably, in step b, the conditions of the sterilization treatment are as follows: the sterilization temperature is 120-125 ℃, and the sterilization time is 25-35 min.

And the bran coat is sterilized, so that other bacteria are not introduced in the later process, the activity of the acinetobacter is further influenced, and even the effect of the microbial inoculum on treating nitrogen-containing sewage is influenced.

Preferably, in the step b, the drying temperature is 30-40 ℃.

The preferable drying temperature can remove the excessive water in the immobilized bacteria agent and does not influence the activity of the acinetobacter.

Preferably, in step b, the mixing condition is stirring at 100-300rpm for 8-12 min.

The preferred mixing conditions are such that the bacterial suspension and carrier are well mixed until the carrier material is wet, loose and non-caking, non-wall sticking.

Preferably, the invention also provides application of the immobilized microbial agent in the field of nitrogen-containing sewage treatment.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a graph showing denitrification performance of an immobilized fungicide provided in example 1 of the present invention.

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 following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The following examples are provided to better illustrate the embodiments of the present invention.

Example 1

The embodiment of the invention provides an immobilized microbial inoculum, which comprises acinetobacter and bran coat, wherein the acinetobacter is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation number of the acinetobacter is CGMCC No. 16620; the water absorption rate of the bran coat is 40%.

The mass ratio of the bran coat to the bacteria suspension of the acinetobacter is 5:2, and the acinetobacter in the bacteria suspension is in a logarithmic growth phase; the OD600 of the bacterial suspension was 0.613.

The preparation method of the immobilized microbial inoculum comprises the following steps:

step a, culturing the Acinetobacter suspension to a logarithmic growth phase;

and b, sterilizing the bran coat at 121 ℃ for 30min, stirring the bran coat and the bacterial suspension at a speed of 200rpm under an aseptic condition for 10min, uniformly mixing until the bran coat is wet, loose, free from caking and wall sticking, and drying at 35 ℃ to obtain the immobilized microbial inoculum.

Example 2

The embodiment of the invention provides an immobilized microbial inoculum, which comprises acinetobacter and bran coat, wherein the acinetobacter is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation number of the acinetobacter is CGMCC No. 16620; the water absorption of the bran coat is 45%.

The mass ratio of the bran coat to the bacteria suspension of the acinetobacter is 4.5:2.5, and the acinetobacter in the bacteria suspension is in a logarithmic growth phase; the OD600 of the bacterial suspension was 0.63.

step a, culturing the Acinetobacter suspension to a logarithmic growth phase;

and b, sterilizing the bran coat at 125 ℃ for 25min, stirring the bran coat and the bacterial suspension under an aseptic condition at the speed of 300rpm for 8min, uniformly mixing until the bran coat is wet, loose, free from caking and wall sticking, and drying at 40 ℃ to obtain the immobilized microbial inoculum.

Example 3

The embodiment of the invention provides an immobilized microbial inoculum, which comprises acinetobacter and bran coat, wherein the acinetobacter is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation number of the acinetobacter is CGMCC No. 16620; the water absorption of the bran coat is 35%.

The mass ratio of the bran coat to the bacteria suspension of the acinetobacter is 5.5:1.5, and the acinetobacter in the bacteria suspension is in a logarithmic growth phase; the OD600 of the bacterial suspension was 0.605.

step a, culturing the Acinetobacter suspension to a logarithmic growth phase;

and b, sterilizing the bran coat at 120 ℃ for 35min, stirring the bran coat and the bacterial suspension under an aseptic condition at a speed of 100rpm for 12min, uniformly mixing until the bran coat is wet, loose, free from caking and wall sticking, and drying at 30 ℃ to obtain the immobilized microbial inoculum.

Example 4

The mass ratio of the bran coat to the bacteria suspension of the acinetobacter is 5:2, and the acinetobacter in the bacteria suspension is in a logarithmic growth phase; the OD600 of the bacterial suspension was 0.153, i.e., the bacterial suspension of example 1 was diluted 1/4 times the original OD 600.

The preparation method of the immobilized microbial inoculum is also the same as that of the example 1 and is not repeated.

Example 5

The comparative example provides an immobilized microbial inoculum, which comprises acinetobacter and bran coat, wherein the acinetobacter is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation number of the acinetobacter is CGMCC NO. 16620; the water absorption rate of the bran coat is 40%.

The mass ratio of the bran coat to the bacteria suspension of the acinetobacter is 5:2, and the OD600 of the bacteria suspension is 1.226, namely, the bacteria suspension in the embodiment 1 is concentrated to be 2 times of the original OD 600.

In order to better illustrate the technical solution of the present invention, further comparison is made below by means of a comparative example and an example of the present invention.

Comparative example 1

The comparative example provides an immobilized microbial inoculum, which comprises acinetobacter and zeolite, wherein the acinetobacter is preserved in the China general microbiological culture Collection center, and the preservation number of the acinetobacter is CGMCC NO. 16620; the water absorption of the zeolite was 40%.

The mass ratio of the zeolite to the acinetobacter bacterial suspension is 5:2, and the OD600 of the bacterial suspension is 2.453, i.e. the bacterial suspension in example 1 is concentrated to 4 times of the original OD 600.

Comparative example 2

The comparative example provides an immobilized microbial inoculum, which comprises acinetobacter and activated carbon, wherein the acinetobacter is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation number of the acinetobacter is CGMCC NO. 16620; the water absorption of the activated carbon was 75%.

The mass ratio of the activated carbon to the bacteria suspension of acinetobacter is 5:2, and the OD600 of the bacteria suspension is 2.453, i.e. the bacteria suspension in example 1 is concentrated to 4 times of the original OD 600.

In order to better illustrate the characteristics of the immobilized bacteria agents provided in the examples of the present invention, the immobilized bacteria agents prepared in examples 1 to 5 and comparative examples 1 to 2 were subjected to performance tests.

Test example 1

150mL of the culture medium was added to 7 flasks, and sterilized at 120 ℃ for 30 min. Then, the immobilized bactericides prepared in examples 1 to 5 and comparative examples 1 to 2 were added into the conical flasks, the added amount of the bactericides was 0.5g by dry weight, the degradation effect on nitrate nitrogen was tested, the degradation efficiency is shown in the following table 1, and the degradation chart of example 1 is shown in fig. 1.

TABLE 1 degradation efficiency

	Initial concentration of nitrate and nitrogen	Time of denitrification	Removal rate of nitro and nitrogen
				Example 1	100mg/L	16h	100％
Example 2	100mg/L	16.1h	100％
				Example 3	100mg/L	16.1h	100％
Example 4	100mg/L	18h	100％
				Example 5	100mg/L	14h	100％
Comparative example 1	100mg/L	20h	35％
				Comparative example 2	100mg/L	20h	5％

As can be seen from Table 1, the immobilized microbial inoculum provided by the embodiments 1-5 of the invention can completely degrade nitrate nitrogen with a concentration of 100mg/L within 18h, wherein the degradation rate of the embodiments 1-3 is better, the nitrate nitrogen can be completely degraded within about 16h, the immobilized microbial inoculum of the embodiments 5 is more excellent, and the nitrate nitrogen can be completely degraded within 14 h. The degradation effect of the immobilized bacteria agent prepared in the comparative examples 1-2 is poor, and the removal rate of nitrol in 20h is only 35% and 5%.

As can be seen from FIG. 1, the immobilized microbial inoculum prepared in example 1 completely degrades 100mg/L of nitrate nitrogen within 16h under aerobic conditions, and completely degrades accumulated nitrite nitrogen, thereby showing good denitrification effect. Unlike free bacteria, the immobilized bacteria prepared by the adsorption method can provide an aerobic and anoxic microenvironment for microorganisms, which is beneficial to the growth of the bacteria and the performance of denitrification and denitrification of the bacteria, and the immobilized bacteria prepared in examples 2 to 5 can completely degrade nitrate nitrogen and nitrite nitrogen within corresponding time.

It can be seen from the combination of examples 1 and 4 to 5 that the denitrification time for achieving a nitrate nitrogen removal rate of 100% is shortened as the concentration of acinetobacter in the immobilized microbial agent is increased.

In comparative examples 1-2, although the concentration of the bacterial suspension used was 4 times that of example 1, the removal rate of nitrol was much lower than that of example 1, and even lower than that of example 4, i.e., the degradation effect of the immobilized bacteria agent using the bacterial suspension concentration 4 times that of comparative examples 1-2 was much lower than that of example 5 using the bacterial suspension concentration 1/4 times.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. An immobilized microbial inoculum, which is characterized in that: the culture medium comprises acinetobacter and an immobilized carrier, wherein the acinetobacter is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number of the acinetobacter is CGMCC NO.16620, and the immobilized carrier is bran.

2. The immobilized inoculant according to claim 1, wherein: the nucleotide sequence of the 16S rDNA of the acinetobacter has a sequence shown as SEQ ID NO. 1.

3. The immobilized inoculant according to claim 1, wherein: the immobilized microbial agent adopts an adsorption method to load the thallus in the bacteria suspension of the acinetobacter onto the immobilized carrier.

4. The immobilized inoculant according to claim 3, wherein: the mass ratio of the immobilized carrier to the bacteria suspension of the acinetobacter is 4.5-5.5: 1.5-2.5.

5. The immobilized inoculant according to claim 3, wherein: the bacterial suspension is obtained by culturing the acinetobacter to logarithmic growth phase, and the OD600 of the bacterial suspension is 0.60-0.63.

6. A method for producing the immobilized fungicide according to any one of claims 1 to 5, characterized in that: at least comprises the following steps:

step a, culturing the acinetobacter to obtain a bacterial suspension;

7. The method for preparing an immobilized microbial inoculum according to claim 6, wherein: in the step b, the conditions of the sterilization treatment are as follows: the sterilization temperature is 120-125 ℃, and the sterilization time is 25-35 min.

8. The method for preparing an immobilized microbial inoculum according to claim 6, wherein: in the step b, the drying temperature is 30-40 ℃.

9. The method for preparing an immobilized microbial inoculum according to claim 6, wherein: in the step b, the mixing condition is stirring for 8-12min under the condition of 100-300 rpm.

10. The use of the immobilized microbial agent of any one of claims 1 to 5 in the field of nitrogen-containing wastewater treatment.