CN107475121A - The prevention and controls of harmful bacteria in a kind of microalga cultivation process - Google Patents

Abstract

The present invention relates to a kind of prevention and controls of harmful bacteria in both culturing microalgae technical field, more particularly to microalga cultivation process.Specific aim improvement can be carried out to there is evil bacterium in microalga cultivation process in time.The prevention and controls of harmful bacteria in a kind of microalga cultivation process, including：Step 1) determines the harmful bacteria that the microalgae has polluted, and the physicochemical property determined by acquisition corresponding to harmful bacteria in microalgae carries out breeding process；Step 2) is cultivated to the microalgae, and in breeding process, the algae solution of the microalgae is monitored, and judges the physicochemical property for whether having corresponding to identified harmful bacteria in the algae solution；If it is determined that having the physicochemical property corresponding to identified harmful bacteria in the algae solution, then, physicochemical property of the step 3) determined by corresponding to harmful bacteria, specific aim improvement is carried out to the harmful bacteria.The embodiment of the present invention is used for both culturing microalgae.

Description

A method for preventing and controlling harmful bacteria in the process of microalgae cultivation

technical field

The invention relates to the technical field of microalgae cultivation, in particular to a method for preventing and controlling harmful bacteria in the microalgae cultivation process.

Background technique

At present, global economic development is constrained by energy shortage. In order to achieve sustainable economic development, microalgae biomass energy has received extensive attention as a renewable energy source. However, the existing large-scale cultivation of algae is usually carried out in open ponds or bioreactors. During the cultivation process, especially in summer, a series of pollution may occur, such as: bacterial pollution, protozoan pollution, etc. Among them, bacterial pollution will occur first in the process of microalgae pollution, and some types of bacteria will reduce the yield of microalgae, and even cause large-scale cultivation to completely collapse within a few days, or even day and night, bringing serious damage to the large-scale cultivation of algae. to great harm.

In order to prevent bacterial contamination during the large-scale cultivation of algae, at present, the culture medium and reactors are usually sterilized in the preparation period before cultivation, so as to avoid microalgae from contaminating bacteria during the cultivation process, and the existing scale Most of the chemical culture adopts open or semi-open culture. Bacterial pollution cannot be completely avoided during the breeding process, and there is no technology to distinguish harmful bacteria from harmless bacteria after pollution occurs. It can only be affected when the growth of microalgae has been affected At this time, the dose of fungicide used is relatively large, which will have a more adverse impact on microalgae that are not growing well.

Therefore, it is urgent to seek a method for early treatment of harmful bacteria in the microalgae culture process, targeted treatment of harmful bacteria, avoiding the decline in microalgae production caused by untimely treatment of harmful bacteria, and the inability to treat harmful bacteria. The reduction of microalgae production caused by targeted treatment occurs.

Contents of the invention

The main purpose of the present invention is to provide a treatment method for harmful bacteria in the process of microalgae cultivation. By monitoring the harmful bacteria in the process of microalgae cultivation in real time, the harmful bacteria that appear in the process of microalgae cultivation can be targeted in time. Treatment, so as to avoid the decrease of microalgae production caused by untimely treatment of harmful bacteria, and the decrease of microalgae production caused by inability to carry out targeted treatment of harmful bacteria.

To achieve the above object, the present invention adopts the following technical solutions:

The embodiment of the present invention provides a method for preventing and controlling harmful bacteria in the microalgae cultivation process, comprising:

Step 1) During the microalgae cultivation process, determine the harmful bacteria that the microalgae has polluted, and obtain the physical and chemical characteristics corresponding to the determined harmful bacteria;

Step 2) cultivating the microalgae, and monitoring the algae liquid of the microalgae during the cultivation process, and judging whether the algae liquid has the physical and chemical characteristics corresponding to the determined harmful bacteria;

If it is determined that the algae liquid has the corresponding physical and chemical characteristics of the determined harmful bacteria, then,

Step 3) According to the physical and chemical characteristics corresponding to the determined harmful bacteria, carry out targeted treatment on the harmful bacteria.

Optionally, the method also includes:

When it is monitored that the number of bacteria in the algae fluid of the microalgae increases, or, after the step 3), the frequency of monitoring is increased.

Optionally, during the cultivation of microalgae, it is determined that the microalgae has been polluted by harmful bacteria; specifically including:

Cultivate the microalgae, and when the microalgae is contaminated with bacteria, and/or, when the bioincrease of the microalgae begins to decrease, the algae liquid of the microalgae is sampled, and the algae liquid in the algae liquid is Bacteria that emerged were colonized;

Bacteria from different colonies obtained from the culture were co-cultivated with sterile microalgae in different experimental groups. If the biomass of the sterile microalgae in the i-th experimental group decreased during the cultivation process, the color of the algal cells changed. Whitening or yellowing and at least one phenomenon in the flocculation of algal cells, then it is determined that the bacteria in the i-th experimental group are harmful bacteria that have been polluted by the microalgae, wherein, i is a natural number greater than or equal to 1, and the The i-th experimental group is any one of all experimental groups.

Optionally, obtain the physical and chemical characteristics corresponding to the identified harmful bacteria; specifically include:

Observing the colony morphology corresponding to each of the identified harmful bacteria to obtain the physicochemical characteristics corresponding to each harmful bacteria; or,

Perform Gram staining on each of the identified harmful bacteria, and observe the cell morphology and color of each harmful bacteria after Gram staining, so as to obtain the corresponding physical and chemical characteristics of each harmful bacteria; or,

The DNA sequence of each identified harmful bacterium is determined separately, and the genus of each harmful bacterium is determined according to the DNA sequence corresponding to each harmful bacterium, so as to obtain the corresponding physical and chemical characteristics of each harmful bacterium.

Optionally, the genus to which each harmful bacterium belongs can be determined according to the DNA sequence corresponding to each harmful bacterium and querying the NCBI database.

Optionally, before the bacteria from different colonies obtained by the culture are cultured together with the sterile microalgae in different experimental groups, the method also includes:

Carry out DNA sequence determination to the bacteria from different colonies, and determine whether the bacteria from different colonies are the same bacteria according to the DNA sequence determination results, to screen out the different bacterial species that are co-cultivated with sterile microalgae in all colonies, and According to the different strains screened out, determine the number of experimental groups for co-culturing bacteria from different colonies and the sterile microalgae.

Optionally, monitor the algae fluid of the microalgae, and determine whether the algae fluid has the physical and chemical characteristics corresponding to the identified harmful bacteria; specifically include:

Sampling the algal fluid of the microalgae, and performing colony culture on the bacteria contaminated by the algae fluid, and comparing the colony morphology of each colony obtained from the culture with the colony morphology corresponding to the determined harmful bacteria; or ,

Sampling the algae fluid of the microalgae, and carrying out Gram staining to the bacteria in the algae fluid, and comparing the cell morphology and the color presented by each bacterium after Gram staining with the determined harmful bacteria for comparison; or,

Sampling the algal liquid of the microalgae, and establishing a phylogenetic tree for the bacteria in the algal liquid, designing specific primers for the 16SDNA sequence of the determined harmful bacteria according to the established phylogenetic tree, and designing specific primers according to the designed phylogenetic tree. Specific primers are used to track the identified harmful bacteria by PCR method.

Optionally, the identified harmful bacteria are tagged and tracked by means of fluorescent quantitative PCR.

Optionally, the step 3) specifically includes:

According to the physical and chemical characteristics corresponding to the determined harmful bacteria, judge the acid and alkali resistance of the determined harmful bacteria, and the difference between the acid and alkali resistance of the harmful bacteria and the cultured microalgae, by adjusting the pH to remediate the identified harmful bacteria; or,

Directly use the appropriate dose of disinfectant to kill the identified harmful bacteria.

The embodiment of the present invention provides a method for preventing and controlling harmful bacteria in the process of microalgae cultivation, by determining in advance the harmful bacteria that have been contaminated by the microalgae during the algae cultivation process, and correspondingly obtaining the Physicochemical properties, in this way, by monitoring the algae liquid of the microalgae in real time during the process of cultivating the microalgae, judging whether the algae liquid has the physical and chemical properties corresponding to the determined harmful bacteria, can Real-time monitoring of the harmful bacteria in the bacteria appearing in the algae liquid, so that when the physical and chemical characteristics of the determined harmful bacteria are monitored in the algae liquid, according to the corresponding physical and chemical characteristics of the determined harmful bacteria characteristics, the targeted treatment of the harmful bacteria can avoid the decrease in the production of microalgae caused by the untimely treatment of harmful bacteria, and the decrease in the production of microalgae caused by the inability to carry out targeted treatment of harmful bacteria, thereby It can carry out early treatment of harmful bacteria and maximize the production of microalgae.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the description of the embodiments. Obviously, the accompanying drawings in the following description are only of the present invention. For some embodiments, those skilled in the art can also obtain other drawings based on these drawings without creative efforts.

1 is a schematic flow diagram of a method for preventing and controlling harmful bacteria in a microalgae cultivation process provided by an embodiment of the present invention;

Fig. 2 is the sensitivity curve diagram of a kind of fluorescent quantitative PCR with 100-fold diluted harmful bacterial genome as template provided by the embodiment of the present invention;

Fig. 3 is a graph of the specificity analysis of a designed specific primer provided by the embodiment of the present invention;

Fig. 4 is a graph of fluorescent quantitative PCR detection results on the day when harmful bacteria were detected in the experimental group and the control group provided by the embodiment of the present invention.

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The embodiment of the present invention provides a method for preventing and controlling harmful bacteria in the process of microalgae cultivation, as shown in Figure 1, including:

Step 1) During the microalgae cultivation process, determine the harmful bacteria that the microalgae has polluted, and obtain the physical and chemical characteristics corresponding to the determined harmful bacteria;

Step 2) cultivating the microalgae, and monitoring the algae liquid of the microalgae during the cultivation process, and judging whether the algae liquid has the physical and chemical characteristics corresponding to the determined harmful bacteria;

If it is determined that the algae liquid has the corresponding physical and chemical characteristics of the determined harmful bacteria, then,

Step 3) According to the physical and chemical characteristics corresponding to the determined harmful bacteria, carry out targeted treatment on the harmful bacteria.

The physical and chemical properties corresponding to harmful bacteria refer to the physical and chemical characteristics of harmful bacteria.

The embodiment of the present invention provides a method for preventing and controlling harmful bacteria in the microalgae culture process, by determining in advance the harmful bacteria that the microalgae has polluted in the microalgae culture process, and correspondingly obtaining the Physicochemical properties, in this way, by monitoring the algae liquid of the microalgae in real time during the process of cultivating the microalgae, judging whether the algae liquid has the physical and chemical properties corresponding to the determined harmful bacteria, can Real-time monitoring of the harmful bacteria in the bacteria appearing in the algae liquid, so that when the physical and chemical characteristics of the determined harmful bacteria are monitored in the algae liquid, according to the corresponding physical and chemical characteristics of the determined harmful bacteria characteristics, the targeted treatment of the harmful bacteria can avoid the decrease in the production of microalgae caused by the untimely treatment of harmful bacteria, and the decrease in the production of microalgae caused by the inability to carry out targeted treatment of harmful bacteria, thereby It can carry out early treatment of harmful bacteria and maximize the production of microalgae.

In an embodiment of the present invention, the method also includes:

When it is monitored that the number of bacteria in the algae fluid of the microalgae increases, or, after the step 3), the frequency of monitoring is increased.

The monitoring frequency of harmful bacteria can be increased, so that harmful bacteria that may appear in the breeding process can be treated in time.

In yet another embodiment of the present invention, during the cultivation of microalgae, it is determined that the harmful bacteria that have been contaminated by the microalgae; specifically include:

Cultivate the microalgae, and when the microalgae is contaminated with bacteria, and/or, when the bioincrease of the microalgae begins to decrease, the algae liquid of the microalgae is sampled, and the algae liquid in the algae liquid is Bacteria that emerged were colonized;

Bacteria from different colonies obtained from the culture were co-cultivated with sterile microalgae in different experimental groups. If the biomass of the sterile microalgae in the i-th experimental group decreased during the cultivation process, the color of the algal cells changed. Whitening or yellowing and at least one phenomenon in the flocculation of algal cells, then it is determined that the bacteria in the i-th experimental group are harmful bacteria that have been polluted by the microalgae, wherein, i is a natural number greater than or equal to 1, and the The i-th experimental group is any one of all experimental groups.

Among them, a colony consists of a single bacterial cell growing and multiplying on the surface or inside of a suitable solid medium to a certain extent, forming a sub-cell community visible to the naked eye.

In the embodiment of the present invention, when the microalgae is contaminated with bacteria, it means that there is obvious bacterial contamination through microscope observation, and when the biological increase of the microalgae begins to decrease, it means that the microalgae is polluted. At this time, by sampling the algae liquid of microalgae and carrying out colony culture to the bacteria in the algae liquid, the colonies of all bacteria in the algae liquid can be obtained, and then the bacteria after the colony culture are compared with those without Bacteria and microalgae are co-cultivated, and it is possible to determine whether there are harmful bacteria in the bacteria contaminated by the microalgae, that is, when the sterile microalgae in a certain experimental group does not grow, the algal cells are obviously white and yellow and the microalgae appear. When there is at least one phenomenon in the obvious flocculation phenomenon, it can be determined that the bacteria in the experimental group are harmful bacteria. Exemplary, when the number of bacterial colonies obtained by cultivating is 3, the bacteria from the 3 bacterial colonies are respectively in the The first experimental group, the second experimental group and the third experimental group are cultured together with sterile microalgae, wherein, when the algal cells in the second experimental group appear to turn white and yellow, it means that the algal cells in the second experimental group Bacteria are harmful bacteria. Here, the first, second, and third names are used to distinguish the experimental groups, and do not limit the setting order and specific setting methods of the experimental groups.

Usually, the bacteria from different colonies are bacteria of different strains, however, in the process of coating the algae liquid, different individuals of the same bacteria may be coated on different positions on the solid medium, so there will be form different colonies.

Therefore, in a preferred embodiment of the present invention, before the bacteria from different colonies obtained by cultivating are respectively cultured with sterile microalgae in different experimental groups, the method also includes:

Carry out DNA sequence determination to the bacteria from different colonies, and determine whether the bacteria from different colonies are the same bacteria according to the DNA sequence determination results, to screen out the different bacterial species that are co-cultivated with sterile microalgae in all colonies, and According to the different strains screened out, determine the number of experimental groups for co-culturing bacteria from different colonies and the sterile microalgae.

In the embodiment of the present invention, by performing DNA sequence determination on bacteria from different colonies, and according to the results of DNA sequence determination, it can be determined whether the bacteria from different colonies are the same bacteria, so that it can be determined that all the colonies contain a total of several different bacteria. Different strains and sterile microalgae are cultured together, which can avoid repeated setting of experimental groups for the same strain and reduce the number of experimental groups. Exemplarily, when the number of bacterium colonies obtained by cultivating is 5, for the convenience of description, these 5 different bacterium colonies will be respectively recorded as the first bacterium colony, the second bacterium colony, the third bacterium colony, the fourth bacterium colony and the fifth bacterium colony in the future, respectively Carry out DNA sequence determination to the bacteria of these 5 different colonies, when it is determined that the first and fourth bacterial colonies are the same bacterium, and the third and fifth bacterial colonies are the same bacterium, then it can be determined that they are from these 5 different bacterial colonies There are three different bacterial species in the bacterium, at this moment, just can determine that the experimental group number that the bacterium from different bacterial colony and described sterile microalgae are cultured together is three groups, promptly the first experimental group can be from the first Bacteria in any one of the bacterium colony and the fourth bacterium colony, the second experimental group can be bacteria from the second bacterium colony, and the third experimental group can be bacteria from any one of the third bacterium colony and the fifth bacterium colony , so as to avoid repeated setting of the experimental group for co-culturing with sterile microalgae for the first bacterial colony and the fourth bacterial colony for the same bacterium, the number of experimental groups can be reduced to 3, reducing the setting of the experimental group. Here, the first, second, and third names are used to distinguish the experimental groups, and do not limit the setting order and specific setting methods of the experimental groups.

Wherein, before DNA sequence determination is carried out to the bacteria from different colonies, the method also includes:

DNA was extracted from bacteria from different colonies, and PCR (Polymerase Chain Reaction, polymerase chain reaction) amplification was performed using bacterial 16S universal primers.

PCR (Polymerase Chain Reaction, Polymerase Chain Reaction) uses DNA to denature in vitro at a high temperature of 95°C and it will become a single strand. Then adjust the temperature to the optimum reaction temperature of DNA polymerase (about 72°C), and DNA polymerase synthesizes complementary chain along the direction from phosphoric acid to five-carbon sugar (5'-3'). It is a molecular biology technique used to amplify specific DNA fragments.

In yet another embodiment of the present invention, the physical and chemical characteristics corresponding to the determined harmful bacteria are obtained; specifically include:

Observe the colony morphology corresponding to each of the identified harmful bacteria, so as to obtain the corresponding physical and chemical characteristics of each harmful bacteria.

Among them, the colony morphology includes the size, shape, edge, luster, texture, color and degree of transparency of the colony. Each kind of bacteria forms fixed colony characteristics under certain conditions.

By observing the colony form corresponding to each harmful bacterium, the colony form corresponding to each harmful bacterium can be distinguished from the colony form of other bacteria, so that the physical and chemical characteristics corresponding to each harmful bacterium can be obtained. Exemplarily, when all bacteria are colonized and determined to be a harmful bacterium, if the colony corresponding to this bacterium is a small round red colony, and the colony is moist, sticky, and easy to provoke , and the colonies corresponding to the rest of the bacteria are, and the colonies are dry and wrinkled, then the physical and chemical characteristics corresponding to the determined harmful bacteria can be obtained as small round red colonies, and the colonies are moist, sticky, and easy to provoke.

Alternatively, perform Gram staining on each of the identified harmful bacteria, and observe the cell morphology and color of each harmful bacteria after Gram staining, so as to obtain the corresponding physical and chemical characteristics of each harmful bacteria;

Gram staining is a differential staining method widely used in bacteriology. Unstained bacteria are extremely difficult to observe under a microscope because of the small difference in refractive index between them and the surrounding environment. After staining, the bacteria are in sharp contrast with the environment, and the shape, arrangement and certain structural features of the bacteria can be clearly observed, which can be used for classification and identification. Exemplarily, when it is determined that a bacterium in all bacteria is a harmful bacterium, by carrying out Gram staining to this bacterium, the shape (such as rod shape, spherical shape, spiral shape, etc.) of this bacterium can be observed under a microscope. ), and the color displayed by this bacterium after Gram staining, compared with the shape of other bacteria and Gram-negative and positive, the physical and chemical characteristics corresponding to the determined harmful bacteria can be obtained as Gram-positive Or Gram-negative rods, or cocci, or spirochetes.

Alternatively, the DNA sequence of each identified harmful bacterium is determined, and the genus of each harmful bacterium is determined according to the DNA sequence corresponding to each harmful bacterium, so as to obtain the corresponding physical and chemical characteristics of each harmful bacterium.

By performing DNA sequence determination on the determined harmful bacteria, and comparing the DNA sequence determination results with known DNA sequences, the genus to which the determined harmful bacteria belongs can be determined, so that according to the genus to which the determined harmful bacteria belong, the commonality, to obtain the physical and chemical properties corresponding to the identified harmful bacteria. Exemplarily, when the genus to which the determined harmful bacteria belongs is Bacillus, and the genus to which other bacteria belong is other genera, it can be obtained that the determined harmful bacteria have the corresponding physical and chemical characteristics of the genus to which they belong, for example: when the determined harmful bacteria When the bacterium is Bacillus, it is a genus of bacteria belonging to the family Bacillus, and it can be obtained that the harmful bacterium is highly resistant to external harmful factors and is a Gram-positive bacterium or the like.

Wherein, in a preferred embodiment of the present invention, the genus to which each harmful bacterium belongs is determined according to the DNA sequence corresponding to each harmful bacterium and queried through the NCBI database.

NCBI (National Center for Biotechnology Information) refers to the National Center for Biotechnology Information in the United States. NCBI's highly trained staff in molecular biology has established a database through sequences submitted by various laboratories and exchanged data with international nucleic acid sequence databases (EMBL and DDBJ). Arrangements with the US Patent and Trademark Office have enabled sequence information on patents to be integrated as well.

In one embodiment of the present invention, the bacteria in the algae fluid of the microalgae are monitored, and it is judged whether the bacteria in the algae fluid have the determined physical and chemical characteristics of harmful bacteria; specifically include:

Sampling the algae fluid of the microalgae, performing colony culture on the bacteria in the algae fluid, and comparing the colony morphology of each colony formed by the culture with the colony morphology corresponding to the determined harmful bacteria.

By carrying out colony culture to the bacteria in the algae liquid during the cultivation process, and comparing the colony morphology of each colony formed with the determined harmful bacteria, the harmful bacteria in the algae liquid can be monitored in real time, for example Specifically, when one of the colonies among all the colonies is found to have the same colony morphology as the determined harmful bacteria through comparison, it can be determined that the algae liquid has the physical and chemical characteristics of the determined harmful bacteria.

Or, sampling the algae fluid of the microalgae, and carrying out Gram staining to the bacteria in the algae fluid, the cell morphology and the presented color of each bacterium after Gram staining and the determined harmful bacteria for comparison.

By Gram staining the bacteria in the algae liquid during the culture process, and comparing the cell morphology and the presented color of each bacteria after Gram staining with the determined harmful bacteria, it is also possible to treat the algae. Real-time monitoring of harmful bacteria in the liquid, for example, when it is found through comparison: the cell shape and color presented by a Gram-stained bacterium are consistent with the determined harmful bacteria, then it can be determined that the The algae liquid has the physical and chemical properties of the identified harmful bacteria.

Or, sampling the algal fluid of the microalgae, and establishing a phylogenetic tree for the bacteria in the algal fluid, designing specific primers for the 16SDNA sequence of the determined harmful bacteria according to the established phylogenetic tree, and designing specific primers according to the established phylogenetic tree. The designed specific primers are used to track the identified harmful bacteria by PCR method.

By establishing a phylogenetic tree for the bacteria in the algae liquid during the cultivation process, and designing specific primers for the 16SDNA sequence of the determined harmful bacteria according to the established phylogenetic tree, by PCR method according to the designed specific primers Tracking the determined harmful bacteria can also monitor the harmful bacteria in the algae liquid in real time.

Preferably, the identified harmful bacteria are tagged and tracked by fluorescent quantitative PCR method. Fluorescent quantitative PCR (realtime fluores-cence quantitative PCR, RTFQ PCR) is a new quantitative test technology introduced by AppliedBiosystems in the United States. It uses fluorescent dyes or fluorescently labeled specific probes to label PCR products. Tracking, real-time online monitoring of the reaction process, combined with the corresponding software can analyze the product and calculate the initial concentration of the sample template to be tested. At the beginning, the probe is bound to any single strand of DNA. During PCR amplification, the 5'-3' end exonuclease activity of Taq enzyme will digest and degrade the probe, making the reporter fluorescent group and quenching fluorescence The group is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, every time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product. The emergence of real-time fluorescence quantitative PCR greatly simplifies the process of quantitative detection, and truly realizes absolute quantification.

In yet another embodiment of the present invention, the step 3) specifically includes:

According to the physical and chemical characteristics corresponding to the determined harmful bacteria, judge the acid and alkali resistance of the determined harmful bacteria, and the difference between the acid and alkali resistance of the harmful bacteria and the microalgae, by adjusting the pH of the algae liquid value to control the identified harmful bacteria.

The identified harmful bacteria can be controlled in a targeted manner, and the damage to the microalgae can be minimized. Exemplarily, when the physical and chemical properties corresponding to the determined harmful bacteria are rod-shaped Gram-negative bacteria (the acid-base tolerance range of the harmful bacteria is between 6.5-7.5) obtained through the above-mentioned Gram staining, And when the corresponding microalgae is Scenedesmus, the pH value of the algae liquid can be adjusted to 4.0 and kept for a certain period of time to kill the harmful bacteria, and the pH value of the algae liquid can be adjusted after the treatment is completed. To the normal growth range of described Scenedesmus.

Or, directly use a suitable dose of disinfectant to kill the identified harmful bacteria.

It is also possible to carry out targeted control on the identified harmful bacteria and reduce the damage to the microalgae to the greatest extent. Exemplarily, when the determined harmful bacteria are marked and tracked by the above-mentioned fluorescent quantitative PCR method, and the concentration of harmful bacteria in the algal liquid is found to be about ¹⁰³ of the initial concentration, then an appropriate dose can be used according to the concentration of the harmful bacteria A suitable disinfectant is used to kill the harmful bacteria, and a suitable dose of disinfectant refers to the dose that can just kill the harmful bacteria without causing damage to the microalgae.

Wherein, the disinfectant can be sodium hypochlorite.

Hereinafter, the present invention will be described in detail through examples, and the technical effects produced by the present invention will be described in detail through examples and comparison groups.

Example 1

Described embodiment 1 takes outdoor 0.2m ² runway ponds to cultivate Nannochloropsis as experimental group, wherein, in the following breeding process, keep the initial inoculation concentration of Nannochloropsis described in each experimental group to be 3g/ L, the number of culture days is 7 days, the culture medium is seawater culture medium, the light conditions are outdoor natural light, and the microalgae culture conditions are measured by measuring the dry weight every 24h.

1. Identify harmful bacteria

Inoculate Nannochloropsis pseudochloropsis in the outdoor 0.2m ² raceway pool, the initial concentration is 3g/L, take the algae liquid that has obvious bacterial contamination under the microscope, take an appropriate amount of samples, and use sterile water to carry out different concentrations by doubling dilution method. Dilute, adjust the concentration of contaminating bacteria to 200/ml, and use a hemocytometer to observe the bacterial concentration.

Take 200 microliters of the corresponding solid bacterial nutrient plate, and carry out bacterial colony cultivation in an incubator at 33 °C.

Pick the bacteria obtained after the above-mentioned colony culture, and activate the bacteria in each colony, extract the DNA from the activated bacteria, use the bacterial 16S universal primer to perform PCR amplification, and then perform DNA sequencing. According to the measured The DNA sequence was used to classify all the colonies into bacteria, and bacteria of different species were picked and mixed with sterile Nannochloropsis at a ratio of 1:1 for co-cultivation at 33°C, and the bacteria of each group were observed. For the growth of Nannochloropsis, if the microalgae does not grow, or the algal cells obviously turn white and yellow, or produce microalgae flocculation, these bacteria are determined to be harmful bacteria.

Conclusion: A total of 36 colonies were obtained, and 7 different bacterial species were obtained after DNA sequencing. By co-cultivating these 7 different bacterial species with sterile Nannochloropsis respectively, we obtained a bacterium that was incompatible with sterile Pseudochloropsis The algal cells turned white after 4 days of co-cultivation of Nannochloropsis.

2. Determination and verification of specific primers for fluorescent quantitative PCR

A phylogenetic tree was established for all bacteria, and specific primers were designed for 16SDNA of harmful bacteria in comparison with the established phylogenetic tree.

The 100-fold diluted target DNA was used as a template to determine the sensitivity of fluorescent quantitative PCR. Among them, the fluorescent quantitative PCR system was 25 microliters, the fluorescent quantitative PCR enzyme was 12.5 microliters, the primer was 1 microliter, and the template was 2 microliters. Microliter, the reaction conditions of fluorescent quantitative PCR are: first keep at 95°C for 30s, then keep at 95°C for 5s, and keep at 60°C for 15s as a cycle, and cycle 40 times; the results are shown in Figure 2, and it can be seen from Figure 2 that: As the DNA dilution ratio decreases, the sensitivity of fluorescent quantitative PCR is stronger, the number of cycles is smaller, and the detection is easier. Therefore, it can be concluded that the detection signal of fluorescent quantitative PCR is related to the concentration of DNA, the detection method is sensitive and reliable, and can be used for quantitative analysis.

The specificity of the designed specific primers was determined by using DNA from two bacterial liquids with close phylogenetic relationship as templates. Among them, the fluorescent quantitative PCR system is 25 microliters, the fluorescent quantitative PCR enzyme is 12.5 microliters, the primer is 1 microliter, and the template is 2 microliters respectively. The reaction conditions of the fluorescent quantitative PCR are: first keep at 95°C for 30s, and then, Keep at 95°C for 5s, keep at 60°C for 15s as a cycle, and cycle 40 times; the results are shown in Figure 3. From Figure 3, it can be seen that the fluorescence intensity of specific genes is more than 10 ⁶ times that of non-specific genes. Therefore, it can be known that The designed specific primers have very high specificity and can be used for early detection of harmful bacteria.

3. Monitoring and treatment of harmful bacteria in the process of microalgae cultivation

Inoculate Nannochloropsis pseudochloropsis in the outdoor ^0.2m2 raceway pool, the initial concentration is 3g/L, culture Nannochloropsis, and in the cultivation process, according to the specific primers determined above, through the fluorescent quantitative PCR method. Harmful bacteria in the liquid are marked and tracked, and they are used as the experimental group to monitor the harmful bacteria; at the same time, a control group under the same culture conditions is set up to monitor the harmful bacteria through microscope inspection during the culture process.

Through monitoring, it is found that in the process of labeling and tracking harmful bacteria by the method of fluorescence quantitative PCR, the concentration of harmful bacteria after 3 days of cultivation is about ¹⁰³ times of the initial concentration, indicating that the experimental group has emerged after 3 days of cultivation. Harmful bacteria, at this time, use 2ppm sodium hypochlorite to treat harmful bacteria, and when the harmful bacteria are monitored through the control group, bacterial contamination is only observed after 5 days of cultivation. At this time, the production of microalgae has declined. trend, and need to use 10ppm of sodium hypochlorite to control harmful bacteria.

Specifically, the corresponding relationship between the fluorescence intensity and the cycle number of the experimental group and the control group on the day when harmful bacteria were detected is shown in Figure 4. It can be seen that the detection of harmful bacteria in the control group has a certain degree of difference compared with the experimental group. Hysteresis, unable to carry out targeted treatment of harmful bacteria in time, will lead to a decline in the production of microalgae. Among them, the results shown in Table 1 below can be obtained by regularly detecting the dry weight of microalgae in the experimental group and the control group.

Table 1

name Yield of experimental group (g/m ² /day) Output of control group (g/m ² /day) Breeding 1 day 7.61 7.7 Breeding for 2 days 8.1 8.03 Breeding for 3 days 7.92 7.79 Breeding for 4 days 7.88 6.98 Breeding for 5 days 8.06 4.88 Breeding for 6 days 7.87 0.35 Breeding for 7 days 8.13 0.75 average yield 7.94 5.21

It can be seen from Table 1 that the experimental group can detect harmful bacteria in time, and after using a small dose of sodium hypochlorite treatment, the bacteria can be effectively controlled without affecting the production of microalgae, while the control group can detect harmful bacteria. The quantity is already very large, and a large dose of sodium hypochlorite is required for treatment, and the production of microalgae has been affected to a certain extent. After treatment with a large dose of sodium hypochlorite, it will cause a certain degree of damage to the microalgae, making the microalgae The yield dropped sharply, and the average yield of the experimental group was greater than that of the control group.

Example 2

In said embodiment 2, the plate reactor with an outdoor optical path of 10 cm is used to cultivate Scenedesmus as an experimental group, wherein, in the following cultivation process, the initial inoculation concentration of Scenedesmus in each experimental group is kept at 3 g/L, and the cultured Scenedesmus is 3 g/L. The number of days is 7 days, the medium is BG11, the light is outdoor natural light, and the microalgae culture is measured by measuring the dry weight of microalgae every 24 hours.

1. Identify harmful bacteria

The specific method is the same as that in Embodiment 1, and will not be repeated here.

Conclusion: A total of 41 colonies were obtained, and the bacteria in the 41 colonies were co-cultured with the sterile Scenedesmus. It can be concluded that there are two kinds of bacteria that are harmful to Scenedesmus, and one of them can kill the microalgae after 5 days. Blanching, another bacterium can flocculate the microalgae cells after 4 days.

2. Determination of the physical and chemical characteristics of harmful bacteria

Perform Gram staining on the harmful bacteria identified above, and observe the cell morphology and the color presented corresponding to the harmful bacteria identified above.

Gram staining results: the above two bacteria are rod-shaped Gram-negative bacteria. The tolerance range of PH value is between 6.5-7.5.

3. Monitoring and treatment of harmful bacteria in the process of microalgae cultivation

Scenedesmus was inoculated in a plate reactor with an outdoor light path of 10cm, the initial concentration was 3g/L, and the Scenedesmus was cultured, and the algae liquid was sampled during the cultivation process, and the bacteria in the algae liquid were tested for Gram. Staining, it is used as an experimental group to monitor harmful bacteria; at the same time, a control group under the same culture conditions is set up, and harmful bacteria are monitored by means of microscope inspection during the culture process.

It is found through monitoring that in the process of monitoring harmful bacteria by Gram staining, it is detected that the identified harmful bacteria appear in the algae liquid on the 3rd day of cultivation, at this time, by adjusting the pH value to 4.0 to maintain 0.5h , and then adjust the pH to the normal culture range of microalgae to control harmful bacteria. When monitoring harmful bacteria through the control group, bacterial contamination was only observed on the 5th day of cultivation. At this time, the production of microalgae has shown a downward trend. Harmful bacteria are treated by means of microscopic inspection. When there is no surviving bacteria in the microscopic examination, it is kept for 1.5 hours, and then the method of adjusting the pH to the normal culture range of microalgae is used to control harmful bacteria.

It can be seen that compared with the experimental group, the detection of harmful bacteria has a certain lag in the control group, and the targeted treatment of harmful bacteria cannot be carried out in time, which will lead to a decline in the production of microalgae. Among them, the results shown in Table 2 below can be obtained by regularly detecting the dry weight of microalgae in the experimental group and the control group.

Table 2

name Yield of experimental group (g/m ² /day) Output of control group (g/m ² /day) Breeding 1 day 8.34 8.56 Breeding for 2 days 8.21 8.3 Breeding for 3 days 7.91 7.97 Breeding for 4 days 7.78 6.83 Breeding for 5 days 7.88 4.69 Breeding for 6 days 7.79 0.36 Breeding for 7 days 8.02 0.65 average yield 7.99 5.34

It can be seen from Table 2 that the experimental group can detect harmful bacteria in time, and use the method of lowering the pH for a short time for treatment, so that the bacteria can be effectively controlled without affecting the production of microalgae, while in the control group, when harmful bacteria are detected, the bacteria The quantity is already very large, and the method of lowering the pH for a long time is required for treatment, and the production of microalgae has been affected to a certain extent. After treatment by the method of lowering the pH for a long time, it will cause a certain degree of damage to the microalgae. As a result, the yield of microalgae also decreased sharply, and the average yield of the experimental group was greater than that of the control group.

Example 3

Described embodiment 3 takes outdoor 2m ² runway pond culture chlorella as experimental group, wherein, in following culture process, keep the initial inoculum concentration of described chlorella in each experimental group to be 3g/L, culture days average For 7 days, BG11 was used as the medium, the light was outdoor natural light, and the microalgae culture was measured by measuring the dry weight of microalgae every 24 hours.

1. Identify harmful bacteria

The specific method is the same as that in Embodiment 1, and will not be repeated here.

Conclusion: A total of 35 colonies were obtained, and DNA sequencing was carried out to obtain 5 different types of bacteria. The 5 different types of bacteria were co-cultured with sterile Chlorella. Algae are harmful and can turn algae cells white after 4 days.

2. Determination of the physical and chemical characteristics of harmful bacteria

Observe that the colony form of the above-mentioned harmful bacteria is moist, sticky, easy to stir up, small round red colonies.

3. Monitoring and treatment of harmful bacteria in the process of microalgae cultivation

Inoculate Chlorella in the outdoor ^2m2 raceway pool, the initial concentration is 3g/L, culture Chlorella, and sample the algae liquid during the cultivation process, and carry out colony culture on the bacteria in the algae liquid, and all the The colony is compared with the colony form of the determined harmful bacteria, and it is used as the experimental group to monitor the harmful bacteria; at the same time, a control group under the same culture conditions is set up, and the harmful bacteria are detected by microscope inspection during the culture process. to monitor.

Through monitoring, it was found that in the process of monitoring harmful bacteria through the experimental group, the identified harmful bacteria were detected in the algae liquid on the third day of cultivation. At this time, the harmful bacteria were controlled by using 2ppm sodium hypochlorite . When the harmful bacteria were monitored by the control group, bacterial contamination was observed on the 5th day of cultivation. At this time, the production of microalgae had shown a downward trend, and 10ppm of sodium hypochlorite was needed to control the harmful bacteria.

It can be seen that compared with the experimental group, the detection of harmful bacteria has a certain lag in the control group, and the targeted treatment of harmful bacteria cannot be carried out in time, which will lead to a decline in the production of microalgae. Among them, by regularly detecting the dry weight of microalgae in the experimental group and the control group, the results shown in Table 3 below can be obtained.

table 3

name Yield of experimental group (g/m ² /day) Output of control group (g/m ² /day) Breeding 1 day 8.22 8.21 Breeding for 2 days 8.04 8.13 Breeding for 3 days 7.91 7.86 Breeding for 4 days 6.78 6.73 Breeding for 5 days 6.54 4.58 Breeding for 6 days 7.07 0.53 Breeding for 7 days 7.02 0.47 average yield 7.37 5.22

It can be seen from Table 3 that the experimental group can detect harmful bacteria in time, and after using a small dose of sodium hypochlorite treatment, the bacteria can be effectively controlled without affecting the production of microalgae, while the control group can detect harmful bacteria. The quantity is already very large, and a large dose of sodium hypochlorite is required for treatment, and the production of microalgae has been affected to a certain extent. After treatment with a large dose of sodium hypochlorite, it will cause a certain degree of damage to the microalgae, making the microalgae The yield dropped sharply, and the average yield of the experimental group was greater than that of the control group.

In summary, by determining the harmful bacteria polluted by the microalgae in the process of microalgae cultivation in advance, and correspondingly obtaining the physical and chemical characteristics corresponding to each of the determined harmful bacteria, in this way, by cultivating the microalgae During the process, the algae liquid of the microalgae is monitored in real time to determine whether the algae liquid has the physical and chemical characteristics corresponding to the determined harmful bacteria, and the harmful bacteria in the bacteria that appear in the algae liquid can be detected. Real-time monitoring, in this way, when the physical and chemical characteristics of the determined harmful bacteria are monitored in the algae liquid, the harmful bacteria are targeted according to the corresponding physical and chemical characteristics of the determined harmful bacteria, which can avoid The decrease of microalgae production caused by untimely treatment of harmful bacteria and the decrease of microalgae production caused by the inability to carry out targeted treatment of harmful bacteria occur, so that early treatment of harmful bacteria can be carried out to maximize the improvement of microalgae production. At the same time, compared with the monitoring of harmful bacteria through microscopic examination in the prior art in the breeding process, it overcomes the relatively strong defect of hysteresis through microscopic examination in the prior art, and can be detected in the early stage of pollution Harmful bacteria, avoiding the impact of harmful bacteria on the production of microalgae, and at the same time avoiding the defect that the treatment method caused by the increase in the number of harmful bacteria is easy to cause damage to microalgae.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (9)

1. A method for preventing and treating harmful bacteria in the microalgae culture process, characterized in that, comprising: Step 1) During the microalgae cultivation process, determine the harmful bacteria that the microalgae has polluted, and obtain the physical and chemical characteristics corresponding to the determined harmful bacteria; Step 2) cultivating the microalgae, and monitoring the algae liquid of the microalgae during the cultivation process, and judging whether the algae liquid has the physical and chemical characteristics corresponding to the determined harmful bacteria; If it is determined that the algae liquid has the corresponding physical and chemical characteristics of the determined harmful bacteria, then, Step 3) According to the physical and chemical characteristics corresponding to the determined harmful bacteria, carry out targeted treatment on the harmful bacteria. 2. The control method according to claim 1, characterized in that, The method also includes: When it is monitored that the number of bacteria in the algae fluid of the microalgae increases, or, after the step 3), the frequency of monitoring is increased. 3. The control method according to claim 1, characterized in that, During the microalgae cultivation process, determine the harmful bacteria that the microalgae has polluted; specifically include: Cultivate the microalgae, and when the microalgae is contaminated with bacteria, and/or, when the bioincrease of the microalgae begins to decrease, the algae liquid of the microalgae is sampled, and the algae liquid in the algae liquid is Bacteria that emerged were colonized; Bacteria from different colonies obtained from the culture were co-cultivated with sterile microalgae in different experimental groups. If the biomass of the sterile microalgae in the i-th experimental group decreased during the cultivation process, the color of the algal cells changed. Whitening or yellowing and at least one phenomenon in the flocculation of algal cells, then it is determined that the bacteria in the i-th experimental group are harmful bacteria that have been polluted by the microalgae, wherein, i is a natural number greater than or equal to 1, and the The i-th experimental group is any one of all experimental groups. 4. The control method according to claim 3, characterized in that, Obtain the physicochemical properties corresponding to the identified harmful bacteria; specifically include: Observing the colony morphology corresponding to each of the identified harmful bacteria to obtain the physicochemical characteristics corresponding to each harmful bacteria; or, Perform Gram staining on each of the identified harmful bacteria, and observe the cell morphology and color of each harmful bacteria after Gram staining, so as to obtain the corresponding physical and chemical characteristics of each harmful bacteria; or, The DNA sequence of each identified harmful bacterium is determined separately, and the genus of each harmful bacterium is determined according to the DNA sequence corresponding to each harmful bacterium, so as to obtain the corresponding physical and chemical characteristics of each harmful bacterium. 5. The control method according to claim 4, characterized in that, According to the DNA sequence corresponding to each harmful bacterium, and through the NCBI database query, to determine the genus to which each harmful bacterium belongs. 6. The control method according to claim 3, characterized in that, Before the bacteria from different bacterium colonies obtained by culturing are cultured together with sterile microalgae respectively in different experimental groups, the method also includes: Carry out DNA sequence determination to the bacteria from different colonies, and determine whether the bacteria from different colonies are the same bacteria according to the DNA sequence determination results, to screen out the different bacterial species that are co-cultivated with sterile microalgae in all colonies, and According to the different strains screened out, determine the number of experimental groups for co-culturing bacteria from different colonies and the sterile microalgae. 7. The control method according to any one of claims 1-6, characterized in that, Monitoring the algae liquid of the microalgae, and judging whether the algae liquid has the physical and chemical characteristics corresponding to the identified harmful bacteria; specifically including: Sampling the algal fluid of the microalgae, and performing colony culture on the bacteria contaminated by the algae fluid, and comparing the colony morphology of each colony obtained from the culture with the colony morphology corresponding to the determined harmful bacteria; or , Sampling the algae fluid of the microalgae, and carrying out Gram staining to the bacteria in the algae fluid, and comparing the cell morphology and the color presented by each bacterium after Gram staining with the determined harmful bacteria for comparison; or, Sampling the algal liquid of the microalgae, and establishing a phylogenetic tree for the bacteria in the algal liquid, designing specific primers for the 16SDNA sequence of the determined harmful bacteria according to the established phylogenetic tree, and designing specific primers according to the designed phylogenetic tree. Specific primers are used to track the identified harmful bacteria by PCR method. 8. The control method according to claim 7, characterized in that, The identified harmful bacteria were tagged and tracked by fluorescent quantitative PCR method. 9. The control method according to claim 1, characterized in that, Described step 3) specifically comprises: According to the physical and chemical characteristics corresponding to the determined harmful bacteria, judge the acid and alkali resistance of the determined harmful bacteria, and the difference between the acid and alkali resistance of the harmful bacteria and the cultured microalgae, by adjusting the pH to remediate the identified harmful bacteria; or, Directly use the appropriate dose of disinfectant to kill the identified harmful bacteria.