CN111197000A

CN111197000A - High temperature resistant ammonia oxidizing bacteria culture device and method

Info

Publication number: CN111197000A
Application number: CN202010181604.6A
Authority: CN
Inventors: 厉巍; 马彦蓉; 郭平均; 王浩; 刘勇弟
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2020-03-16
Filing date: 2020-03-16
Publication date: 2020-05-26

Abstract

The invention discloses a high temperature resistant ammonia oxidizing bacteria culture device and a method thereof. In the device, the supporting dish is used for accommodating and placing the culture container; the culture container includes an inner culture dish and an outer culture dish, the inner culture dish and the outer culture dish share the same bottom surface, and an annular accumulation space is formed between the sides of the two; The bottom of the side wall of the petri dish is connected with the bottom surface with a number of communicating holes in the circumferential direction, and the inner cavity of the inner petri dish is communicated with the annular accumulation space through the communicating holes; the bottom of the interior cavity of the inner petri dish is covered with a layer of water-absorbing filter paper; The outer culture dish cover body and the feeding tube are provided with a through hole on the outer culture dish cover body, and the feeding tube extends into the annular storage space after passing through the through hole. The method can use batch feeding to improve the water content and nutrient content of the solid medium, effectively solve the problems of dry cracking caused by long-term cultivation of the traditional solid medium, and prolong the cultivation time of the heat-resistant ammonia oxidizing microorganism.

Description

High-temperature-resistant ammonia oxidizing bacteria culture device and method

Technical Field

The invention belongs to the technical field of environmental microorganism tools, relates to a vessel, and particularly relates to a high-temperature-resistant ammonia oxidizing bacteria culture device.

Background

With the increasingly serious trend of water eutrophication at home and abroad and the high attention of relevant departments to the problem, the reduction of external sources becomes the primary measure for controlling the water eutrophication, so that a large number of sewage treatment plants are built, and sewage is effectively treated by a specific process so as to reduce the input of pollutants into natural water. The effluent discharge of a sewage treatment plant is an important source of nitrogen pollution of water bodies, in order to effectively control and relieve the eutrophication phenomenon of the water bodies, the nitrogen removal requirement of the sewage treatment plant is more and more strict, at present, the sewage treatment plant in China generally requires to execute the first-class A or first-class B standard of pollutant discharge standard of urban sewage treatment plants, wherein the ammonia nitrogen concentration of the effluent needs to be controlled below 5mg/L or 8 mg/L.

At present, biological methods are generally adopted at home and abroad for denitrification, and autotrophic nitrification-heterotrophic denitrification with continuous flow operation of activated sludge method is taken as a mainstream process, and A²The form of/O is represented. In general, a common denitrification process includes two stages, nitrification and denitrification. The main functional microorganism in the nitrification stage is nitrifying bacteria, which are autotrophic microorganisms, slow in growth and easily affected by water quality conditions and environmental factors. The nitration process can be divided into two steps, the first of which is referred to as the ammonia oxidation process, i.e. ammonia (NH)₃) Conversion to Nitrite (NO) by ammonia oxidizing microorganisms₂ ^-) The process of (2); the second step is called nitrite oxidation process, i.e. Nitrite (NO)₂ ^-) Further conversion to Nitrate (NO) by nitrite-oxidizing microorganisms₃ ^-) The process of (1). Considering the characteristics of autotrophic microorganisms such as generation cycle and metabolic type, the ammoxidation process is generally considered to be nitrateThe speed-limiting step of the chemical process influences the smooth operation of the whole denitrification process. Therefore, the enhancement of the research on the functional microorganisms in the ammonia oxidation process is helpful for deepening the understanding of the denitrification process in the sewage treatment.

As functional microorganisms for the ammoxidation process, ammonia oxidizing bacteria are required to promote the ammoxidation process of activated sludge by improving the ammoxidation performance of the ammonia oxidizing bacteria. Since the temperature of part of the industrial wastewater is often higher than 35 ℃ and the optimum growth temperature of the ammonia oxidizing bacteria is about 30 ℃, the ammonia oxidizing performance of the ammonia oxidizing bacteria is weakened or even the ammonia oxidizing bacteria stop growing in an environment exceeding 35 ℃. Meanwhile, the ammonia oxidizing bacteria are autotrophic bacteria, grow slowly, have long multiplication time and take several days. Therefore, it is desirable to select ammonia oxidizing bacteria that can grow faster at temperatures above 35 ℃ and have better efficiency.

At present, the conventional single round box structure is mostly adopted in the commonly used bacterial culture dish, the function is single, more defects exist, the culture medium is easy to dehydrate under the high-temperature condition to cause cracking, and bacteria can not be normally cultured, so that a high-temperature resistant ammonia oxidizing bacteria culture device is needed, and the ammonia oxidizing bacteria can be normally cultured for a long time under the high-temperature condition.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a high-temperature resistant ammonia oxidizing bacteria culture device.

The invention adopts the following specific technical scheme:

a high-temperature resistant ammonia oxidizing bacteria culture device comprises a bearing vessel, a culture container and a feeding cover;

the supporting vessel is used for accommodating and placing a culture container;

the culture container comprises an inner culture dish and an outer culture dish, the inner culture dish and the outer culture dish share the same bottom surface, and an annular accumulation space is formed between the side surfaces of the inner culture dish and the outer culture dish; a plurality of communication holes are circumferentially arranged at the connecting position of the bottom and the bottom of the side wall of the inner culture dish, the inner cavity of the inner culture dish is communicated with the annular accumulation space through the communication holes, and the rest positions of the side wall of the inner culture dish are sealed and watertight; a layer of water-absorbing filter paper is laid at the bottom of the inner cavity of the inner culture dish, and the outer wall of the water-absorbing filter paper is attached to the inner wall of the inner culture dish;

the feed supplement cover comprises an outer culture dish cover body and a feed supplement pipe; the diameter of the outer culture dish cover body is larger than that of the outer culture dish, and the outer culture dish cover body can cover the outer culture dish; the outer culture dish cover body is provided with a through hole, and the feed supplement pipe penetrates through the through hole and then extends into the annular accumulation space.

Preferably, the thickness of the water absorbing filter paper is larger than the height of the communicating hole.

Preferably, the supporting vessel, the culture vessel and the feeding cover are all made of transparent glass.

Preferably, the cross-sectional outer contours of the holding vessel, the culture vessel and the supplement cover are all circular.

Preferably, the diameter of the outer culture dish cover body is 6-8.5 cm, and the height of the outer culture dish cover body is 1.8-2 cm.

Preferably, the through hole is located 5-8 mm away from the edge of the cover body, the inlet of the material supplementing pipe is in a barb form, the material supplementing pipe is changed from thin to thick from the inlet to the outlet, and the diameter of the pipe orifice is gradually increased from 1.5-2 mm to 5-6 mm; the height of the material supplementing pipe is 4-5 cm, the height of the part, located below the outer culture dish cover body, of the material supplementing pipe is 0.8-1 cm, and the height of the part located above the material supplementing pipe is 3.2-4 cm.

Preferably, the diameter of the inner culture dish is 5-7 cm, and the height of the inner culture dish is 1.5-1.8 cm; the diameter of the outer culture dish is 5.5-8 cm, and the height is 1.5-1.8 cm.

Preferably, the communication holes are rectangular holes, the length of each rectangular hole is 1-1.2 cm, the height of each rectangular hole is 0.1-0.2 mm, and the circumferential interval between every two adjacent rectangular holes is 0.2-0.4 cm.

Preferably, the diameter of the bearing dish is 6.5-9 cm, and the height is 2-2.2 cm.

Another object of the present invention is to provide a method for culturing ammonia oxidizing bacteria in the apparatus for culturing high temperature-resistant ammonia oxidizing bacteria according to any one of the above aspects, comprising the steps of:

placing the culture container into the bearing dish in a mode that the opening faces upwards, and then fully spreading the water-absorbing filter paper on the bottom of the inner culture dish; then pouring the culture medium into an inner culture dish, and after the culture medium is cooled and fixed, performing ammonia oxidizing bacteria separation culture on the plane of the culture medium through a coating rod or an inoculating loop; and finally, injecting a layer of culture solution into the annular accumulation space between the inner culture dish and the outer culture dish, covering the supplement cover above the culture container, putting the culture container into an incubator for high-temperature culture, and periodically supplementing the culture solution into the annular accumulation space through the supplement pipe in the culture process, so that the water-absorbing filter paper continuously absorbs the culture solution through capillary action for supplementing water and nutrient components to the culture medium.

Compared with the prior art, the high-temperature-resistant ammonia oxidizing bacteria culture device provided by the invention is designed into a double-layer structure consisting of the inner culture dish and the outer culture dish on the basis of the traditional culture dish, and the water absorption filter paper at the bottom of the inner culture dish is matched with the communication hole, so that the water absorption filter paper continuously absorbs the culture solution in the annular accumulation space between the inner culture dish and the outer culture dish through the capillary action. Therefore, the culture device can continuously supplement water and nutrient components to the culture medium, so that the culture medium is not easy to crack under the high-temperature condition, and the ammonia oxidizing bacteria can obtain longer separation culture time. The culture device is simple in structure and convenient to use, can culture the ammonia oxidizing bacteria at a high temperature for a long time, and is suitable for the culture experiment of the ammonia oxidizing bacteria.

Drawings

FIG. 1 is an exploded view of a culture apparatus for ammonia oxidizing bacteria resistant to high temperatures.

FIG. 2 is a schematic diagram of the overall structure of a high-temperature resistant ammonia oxidizing bacteria culture apparatus.

FIG. 3 is a schematic structural diagram of a support vessel in a high-temperature resistant ammonia oxidizing bacteria culture device.

FIG. 4 is a schematic diagram showing the structure of a culture vessel in a culture container for a high-temperature resistant ammonia oxidizing bacterium.

FIG. 5 is a schematic longitudinal sectional view of a culture vessel in a culture container for a high-temperature resistant ammonia oxidizing bacterium (non-absorbent filter paper).

FIG. 6 is a schematic longitudinal sectional view of a culture vessel in a culture container for a high-temperature resistant ammonia oxidizing bacterium (with a water-absorbing filter paper).

FIG. 7 is a schematic view of a culture vessel in which a high-temperature resistant ammonia oxidizing bacterium is cultured and used.

FIG. 8 is a schematic structural view of a supplement cover in the culture device for the high-temperature resistant ammonia oxidizing bacteria.

The reference numbers in the figures are: the culture device comprises a holding dish 1, a culture container 2, a feeding cover 3, an inner culture dish 201, an outer culture dish 202, a supporting point 203, water-absorbing filter paper 204, a culture medium 205, a culture solution 206, an outer culture dish cover 301 and a feeding tube 302.

Detailed Description

The invention will be further elucidated and described with reference to the drawings and the detailed description. The technical features of the embodiments of the present invention can be combined correspondingly without mutual conflict.

Referring to fig. 1, a preferred embodiment of the present invention provides a culture apparatus for ammonia oxidizing bacteria with high temperature resistance, which comprises a support dish 1, a culture container 2, and a feeding cover 3, wherein the three parts are assembled as shown in fig. 2 when in use, and can be disassembled again after use. In this embodiment, the support dish 1, the culture container 2 and the supplement cover 3 are made of transparent glass, and the cross-sectional outlines of the three are circular, but the specific material and property can be changed according to actual needs, and are not limited.

It should be noted that "high temperature" in the present invention is merely a relative concept, and is a temperature higher than room temperature, which is easy to cause evaporation of water, and does not refer to a certain absolute temperature. Generally, the culture temperature of the ammonia oxidizing bacteria is 35 ℃ or higher.

The support vessel 1 is the basis of the whole culture device, and as shown in FIG. 3, it is a cylindrical vessel without a lid, and the main function is to accommodate the culture vessel 2 and to bear the weight of the culture vessel 2 and the feeding lid 3.

Referring to FIG. 4, the culture container 2 includes an inner culture dish 201 and an outer culture dish 202, and the inner culture dish 201 and the outer culture dish 202 are both in the shape of a non-top cylindrical container and share the same bottom surface. In the embodiment, the two are coaxially arranged, and an annular accumulation space is formed between the side surfaces of the two in a clamping mode. The inner culture dish 201 is a main place for culturing ammonia oxidizing bacteria, and the inside thereof is added with a culture medium according to the need of the test, and then the ammonia oxidizing bacteria are inoculated in the culture medium by a coating rod or an inoculating loop for culturing. However, since the entire culture apparatus is in a high temperature environment, moisture in the culture medium is easily evaporated, the culture medium is dehydrated and cracked, and bacteria cannot be normally cultured. The annular accumulation space serves to accommodate additional culture fluid for supplementing the medium moisture that should be lost at high temperatures. However, the water replenishing channel is not directly penetrated, and capillary water absorption is realized by matching the water absorption filter paper 204 with the communication hole. The black disc in fig. 4 indicates the position of the water absorbing filter paper 204, when the water absorbing filter paper 204 is removed, the structure of the culture container 2 is as shown in fig. 5, the bottom and the bottom surface of the side wall of the inner culture dish 201 are not completely sealed, but are supported on the bottom surface through a plurality of support points 203 along the circumferential direction, a communication hole is formed between any two adjacent support points 203, and the communication holes are uniformly arranged along the circumferential direction of the bottom of the side wall of the inner culture dish 201. Thus, the inner chamber of the inner culture dish 201 communicates with the annular accumulation space through these communication holes, while the remaining positions of the side wall of the inner culture dish 201 are hermetically sealed and watertight. When the water-absorbing filter paper 204 is put into the culture container 2, the structure is shown in fig. 6, and at this time, the water-absorbing filter paper 204 is spread over the bottom of the inner cavity of the inner culture dish 201, and the outer wall of the water-absorbing filter paper 204 should be attached to the inner wall of the inner culture dish 201. In the use state shown in fig. 7, after the culture medium 205 is injected into the inner culture dish 201, the culture medium 205 inside hardly permeates into the annular accumulation space between the inner culture dish 201 and the outer culture dish 202 due to the blocking effect of the water-absorbent filter paper 204, and the thickness of the water-absorbent filter paper 204 should be as large as possible than the height of the communication hole in order to ensure the blocking effect. Then, a layer of culture solution 206 is further injected into the annular accumulation space between the inner culture dish 201 and the outer culture dish 202, and at this time, since the outer wall of the water-absorbing filter paper 204 can directly contact the culture solution 206 outside through the communication hole, it will continuously absorb water and nutrients in the culture solution 206 and transfer them to the culture medium 205 on the upper portion under the siphon action of capillary effect, and uniformly supply nutrients and water to the culture medium 205 for a long period of time. The annular accumulation space between the inner culture dish 201 and the outer culture dish 202 can be periodically replenished with culture solution 206, so that the culture solution 206 in the annular accumulation space is prevented from being exhausted. The depth of the culture solution 206 should not be too high, and is controlled to about 0.5cm, so that the water-absorbing filter paper 204 can be wetted.

The feeding cover 3 is covered above the culture container 2 to reduce water evaporation and the interference of bacteria in the environment. Since the culture solution 206 needs to be supplemented periodically in the present invention, the feeding cap 3 is shown in FIG. 8 and comprises an outer culture dish cover 301 and a feeding tube 302. The outer culture dish cover 301 is a bottomless cylindrical cover, and the inner diameter of the outer culture dish cover 301 should be larger than the outer diameter of the outer culture dish 202 so that it can cover the outer culture dish 202. In addition, the outer diameter of the outer culture dish cover 301 is smaller than the inner diameter of the support dish 1, so that it can be snapped between the support dish 1 and the outer culture dish 202. The outer culture dish cover body 301 is provided with a through hole, and the feed supplement pipe 302 penetrates through the through hole and then extends into the annular accumulation space to supplement the culture solution 206. To reduce the water loss through the feed tube 302, the inlet of the feed tube 302 is designed to be barbed, i.e., the culture fluid 206 needs to flow upward a certain distance after entering its inlet end, bypassing the elbow and then entering the annular accumulation space downward. The culture medium 206 can be added by injecting a liquid into the inlet of the feed supplement tube 302 using an auxiliary device such as a syringe.

The culture device provided by the invention can adopt fed-batch culture, improves the water content and the nutrient content of the solid culture medium, can effectively solve the problems of dry cracking and the like caused by long-term culture of the traditional solid culture medium, and prolongs the culture time of the heat-resistant ammonia oxidation microorganisms.

In this culture apparatus, the concrete dimensional parameter of support ware 1, culture vessel 2 and feed supplement lid 3 triplex can be adjusted according to the experimental requirement of cultivateing, can design the specification and size of multiple difference, does not influence the realization of the whole function of the device. In general, considering the requirements of a conventional culture experiment, the dimensional parameters can be referred to the following ranges:

the diameter of the outer culture dish cover 301 can be set to 6-8.5 cm, and the height is 1.8-2 cm. The through hole through which the material supplementing pipe 302 penetrates can be formed in the position 5-8 mm away from the edge of the cover body, and the outer portion of the material supplementing pipe 302 is tightly fit with the through hole. The material supplementing pipe 302 preferably adopts a mode of changing from thin to thick from an inlet to an outlet, and the diameter of the pipe orifice is gradually increased from 1.5-2 mm to 5-6 mm. The height of the material supplementing pipe is 4-5 cm, the height of the part, located below the outer culture dish cover body 301, of the material supplementing pipe is 0.8-1 cm, and the height of the part located above the material supplementing pipe is 3.2-4 cm. The diameter of the inner culture dish 201 can be set to be 5-7 cm, and the height is 1.5-1.8 cm; the diameter of the outer culture dish 202 can be set to 5.5-8 cm, and the height is 1.5-1.8 cm. Rectangular holes are preferably adopted in the communication holes, the length of each rectangular hole is 1-1.2 cm, the height of each rectangular hole is 0.1-0.2 mm, and the circumferential interval between every two adjacent rectangular holes is 0.2-0.4 cm. The diameter of the bearing dish 1 can be set to 6.5-9 cm, and the height is 2-2.2 cm. The specific selection of the parameters should meet the requirement for realizing the matching relationship between the components, and can be properly adjusted in the range, and of course, other parameter setting ranges can also be adopted.

Based on the high-temperature resistant ammonia oxidizing bacteria culture device, a high-temperature culture method for ammonia oxidizing bacteria culture can be further provided, and the method comprises the following specific steps:

first, the culture container 2 is placed in the holding dish 1 with the opening facing upward, and the water-absorbent filter paper 204 is spread over the bottom of the inner culture dish 201.

Then, the medium 205 is poured into the inner dish 201, and after the medium 205 is cooled and fixed, the ammonia oxidizing bacteria are isolated and cultured on the medium plane by a coating rod or an inoculating loop.

Finally, a layer of culture solution 206 is injected into the annular accumulation space between the inner culture dish 201 and the outer culture dish 202, the supplement cover 3 is covered above the culture container 2, and then the culture container is placed into an incubator for high-temperature culture, and the specific culture temperature and culture time are determined according to the test requirements.

In the culture process, the height of the culture solution 206 in the annular accumulation space needs to be observed regularly, when the culture solution 206 in the annular accumulation space is too small, the culture solution 206 needs to be supplemented to the annular accumulation space regularly through the material supplementing pipe 302, the water absorption filter paper 204 continuously absorbs the culture solution 206 through capillary action for supplementing water and nutrient components to the culture medium 205, the culture medium 205 is not easy to crack under the high-temperature condition, and the ammonia oxidizing bacteria can be cultured for a longer separation time.

The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.

Claims

1. a high temperature resistant ammonia oxidizing bacteria cultivation device, is characterized in that, comprises supporting dish (1), culture container (2) and feeding cover (3);

The supporting dish (1) is used for accommodating and placing the culture container (2);

The culture container (2) comprises an inner culture dish (201) and an outer culture dish (202), the inner culture dish (201) and the outer culture dish (202) share the same bottom surface, and the side surfaces of the two are sandwiched to form an annular accumulation space; a plurality of communication holes are circumferentially provided at the position where the bottom of the side wall of the inner culture dish (201) meets the bottom surface, and the inner cavity of the inner culture dish (201) is communicated with the annular accumulation space through the communication holes, and the inner culture dish (201) The rest of the side wall of the ) is airtight and watertight; the inner cavity bottom of the inner culture dish (201) is laid with a layer of water-absorbing filter paper (204), and the outer wall of the water-absorbing filter paper (204) fits the inner wall of the inner culture dish (201);

The feeding cover (3) comprises an outer petri dish cover (301) and a feeding tube (302); the diameter of the outer petri dish cover (301) is larger than the diameter of the outer petri dish (202), The outer culture dish (202) can be covered with a through hole; the outer culture dish cover (301) is provided with a through hole, and the feeding pipe (302) extends into the annular storage space after passing through the through hole.

2 . The high temperature resistant ammonia oxidizing bacteria culture device according to claim 1 , wherein the thickness of the water-absorbing filter paper ( 204 ) is greater than the height of the communicating holes. 3 .

3 . The high temperature resistant ammonia oxidizing bacteria culture device according to claim 1 , wherein the supporting dish ( 1 ), the culture container ( 2 ) and the feeding cover ( 3 ) are all made of transparent glass. 4 .

4. The high temperature resistant ammonia oxidizing bacteria culture device according to claim 1, wherein the cross-sectional outer contours of the supporting dish (1), the culture container (2) and the feeding cover (3) are all circular shape.

5 . The high temperature resistant ammonia oxidizing bacteria culture device according to claim 1 , wherein the outer petri dish cover body ( 301 ) has a diameter of 6-8.5 cm and a height of 1.8-2 cm. 6 .

6. The high temperature resistant ammonia oxidizing bacteria culture device according to claim 1, wherein the through hole is located at a distance of 5 to 8 mm from the edge of the cover body, and the inlet of the feeding pipe (302) is in the form of a barb, and the feeding pipe (302) is in the form of a barb. The feeding tube (302) changes from thin to thick from the inlet to the outlet, and the diameter of the nozzle gradually increases from 1.5 to 2 mm to 5 to 6 mm; the height of the feeding tube is 4 to 5 cm, and is located at the lower part of the outer petri dish cover (301). The height is 0.8 to 1 cm, and the height of the upper part is 3.2 to 4 cm.

7. The high temperature resistant ammonia oxidizing bacteria culture device according to claim 1, wherein the inner culture dish (201) has a diameter of 5-7 cm and a height of 1.5-1.8 cm; the outer culture dish (202) ) is 5.5 to 8 cm in diameter and 1.5 to 1.8 cm in height.

8 . The high temperature resistant ammonia oxidizing bacteria culture device according to claim 1 , wherein the communicating hole is a rectangular hole, the length of the rectangular hole is 1-1.2 cm, the height is 0.1-0.2 mm, and the adjacent rectangular holes are 1-1.2 cm in length. 9 . The circumferential interval between them is 0.2 to 0.4 cm.

9 . The high temperature resistant ammonia oxidizing bacteria culture device according to claim 1 , wherein the diameter of the supporting dish ( 1 ) is 6.5-9 cm, and the height is 2-2.2 cm. 10 .

10. A method for culturing ammonia oxidizing bacteria according to any one of claims 1 to 9, wherein the culturing container (2) is placed in the form with the opening facing upwards. In the supporting dish (1), the water-absorbing filter paper (204) is fully spread on the bottom of the inner petri dish (201); then the culture medium (205) is poured into the inner petri dish (201), and the culture is waited for After the base (205) is cooled and fixed, the ammonia oxidizing bacteria are separated and cultured on the medium plane through a coating rod or an inoculation ring; finally, the liquid is injected into the annular accumulation space between the inner culture dish (201) and the outer culture dish (202). A layer of culture liquid (206), after covering the feeding cover (3) above the culture container (2), put it into an incubator for high temperature culture, and during the culture process, the feeding pipe (302) is used to The annular accumulating space regularly replenishes the culture fluid (206), so that the water-absorbing filter paper (204) continuously absorbs the culture fluid (206) through capillary action, and is used to supplement water and nutrients to the culture medium (205), so that the culture medium (205) is kept under high temperature conditions. It is not easy to crack, and ammonia oxidizing bacteria can obtain longer separation and culture time.