CN117866856B - Actinomycetes D18 with algae dissolving function, bacterial liquid, sterile filtrate, preparation method and application - Google Patents
Actinomycetes D18 with algae dissolving function, bacterial liquid, sterile filtrate, preparation method and application Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of environmental water treatment, and in particular discloses actinomycetes D18 with an algae dissolving function, bacterial liquid, sterile filtrate, a preparation method and application, wherein actinomycetes D18 are preserved in China center for type culture Collection in 8 months of 2023, and the preservation number is: cctccc NO: m20231417, classified and named: streptomyces venezuelae D18 and 18. The bacterial liquid and the sterile filtrate prepared by the actinomycetes D18 can be used for algae dissolution and water environment denitrification, and can be used for treating water pollution and preventing and treating water bloom.
Description
Technical Field
The invention relates to the technical field of environmental water treatment, in particular to actinomycetes D18 with an algae dissolving function, bacterial liquid, sterile filtrate, a preparation method and application.
Background
The microcystis aeruginosa in blue algae is a harmful algae commonly existing in a global fresh water environment, and research on effective strategies is an important research topic for coping with the explosion of the microcystis aeruginosa. Therefore, the problem of preventing and controlling cyanobacteria bloom is attracting attention.
At present, the methods for preventing and treating the water bloom mainly comprise three types: physical, chemical and biological methods. The physical algae removal method mainly comprises an adsorption method, an ultrasonic method, a mechanical algae removal method, a shading algae inhibition method and the like, but has limited physical algae removal capacity, large workload, long time consumption, high energy consumption, low efficiency and potential secondary pollution risk, so the method is often used as an auxiliary measure for treating the cyanobacteria bloom. The chemical algae removal method mainly uses metal salt, herbicide and other chemical reagents to kill algae, and has high algae removal efficiency, but poor selectivity and great toxicity to other aquatic organisms; and the metal salt is easy to enrich in the water environment and easy to produce secondary pollution. The biological algae removal method mainly comprises population competition algae inhibition, fish phagocytosis algae removal, microorganism algae removal and the like. The physical and chemical methods are inconvenient for the implementation of algae removal and control in large-area water bodies, and the biological rule can realize the effective control and treatment of the cyanobacteria bloom in large-area water bodies.
Some microorganisms that have a killing effect on cyanobacteria can be used for the control of detrimental algal bloom, including phagostimulants, bacteria, fungi, and protozoa. The microbial algae removal utilizes the natural interaction relation of algae-bacteria in natural ecological environment to control algae and remove algae, has the characteristics of simple method, low cost, quick response, environmental friendliness and the like, and has good prospect in the technical development of water bloom algae treatment. This type of microorganism that kills harmful cyanobacteria or inhibits the growth of cyanobacteria is called algicidal bacteria.
At present, the strain with algae dissolving function is single, and is difficult to meet the requirements on water environment treatment. Therefore, the screening of new strains with algicidal function is significant.
Disclosure of Invention
In order to obtain microorganisms with algae dissolving function, the invention provides actinomycetes D18 with algae dissolving function, bacterial liquid, sterile filtrate, a preparation method and application thereof, and the bacterial liquid and the sterile filtrate prepared by the actinomycetes D18 can be used for algae dissolving and water environment denitrification.
The invention provides actinomycetes D18 with algae dissolving function, wherein actinomycetes D18 are preserved in China center for type culture Collection in the year 2023, 8 and 10, and the preservation number is as follows: cctccc NO: m20231417, classified and named: streptomyces venezuelae D18 and 18.
The invention also provides an extracellular active substance secreted by actinomycetes D18 and having an algicidal function and a denitrification function, wherein the extracellular active substance can be used as an inhibitor of an electron transfer chain in a thylakoid membrane.
The invention also provides a bacterial liquid prepared from the actinomycetes D18.
The invention also provides a preparation method of the bacterial liquid, the D18 bacterial liquid is obtained by inoculating the D18 bacterial strain into a DM culture medium and culturing the strain at 28-30 ℃ and 130-150 rpm until the strain grows to logarithmic phase.
The invention also provides a sterile filtrate prepared from the actinomycetes D18.
The invention also provides a preparation method of the sterile filtrate, which comprises the following steps:
culturing the D18 strain to a logarithmic growth phase to obtain a D18 bacterial liquid;
Centrifuging the D18 bacterial liquid to collect supernatant, and filtering out the bacterial strain by using a sterile filter membrane to obtain sterile filtrate.
Further, the centrifugation conditions are: 8000-10000 r/min, and centrifuging for 10-15 min.
The invention also provides application of the actinomycetes D18, bacterial liquid or the sterile filtrate in water treatment, wherein the bacterial liquid or the sterile filtrate is used for water bloom prevention and treatment.
Further, the actinomycete D18, bacterial liquid or sterile filtrate is used for removing microcystis aeruginosa in water environment.
Further, the actinomycetes D18, bacterial liquid or sterile filtrate is used for denitrification of sewage or wastewater.
Further, the denitrification is to reduce the content of total nitrogen, nitrate, nitrite and ammonia in the water environment.
The invention also provides application of the extracellular active substance in water bloom prevention and treatment.
Compared with the prior art, the invention has the beneficial effects that:
1. The bacterial liquid and the sterile filtrate prepared by the actinomycetes D18 can be used for algae dissolution and water environment denitrification, and can be used for water bloom prevention and treatment. According to the experiment, actinomycetes D18 can secrete an extracellular active substance for algae dissolving. Experiments show that the algae dissolution rates of the D18 bacterial liquid and the sterile filtrate respectively reach 85.0% and 83.6%.
2. The sterile filtrate prepared from the D18 strain provided by the invention can effectively inhibit the generation of allophycocyanin, phycocyanin and phycoerythrin by algal cells. The aseptic filtrate causes oxidative stress of algae cells, and as the stress time is prolonged, the algae cell function metabolism is damaged, and photosynthesis is destroyed, so that the capturing can not be performed efficiently, and the synthesis of carotenoid is indirectly influenced. The addition of the sterile filtrate affects the conversion of carotenoids, and the decrease in pigment content causes a decrease in the fluorescence parameters of the chlorophyll of the photosystem. The decrease in photosynthetic capacity indicates that the autotrophic capacity of algal cells is reduced and cell growth is limited.
The algicidal effect of the sterile filtrate on the algal cells may also act by the following pathways: the aseptic filtrate causes imbalance in ATP production and consumption, resulting in abnormal growth of algal cells; extracellular active substances produced by D18 inhibit the transfer of electrons in the electron transfer chains in thylakoid membranes.
3. Experiments prove that actinomycetes D18 in the invention has good denitrification characteristics, the removal rate of TN can reach 88.1%, and the removal rate of NO 3 - -N can reach 96.05%. The method also shows good denitrification characteristics and algae dissolving effect in practical application.
Biological material preservation information
D18, herein designated actinomycete D18, was deposited at the chinese collection at 2023, 8, 10, under the accession number: cctccc NO: m20231417, the preservation unit address is Chinese, wuhan, university of Wuhan, post code: 430072, class designation: streptomyces venezuelae D18 and 18.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the morphological characteristics of actinomycetes D18 strain and the algicidal effect of D18 bacterial liquid, D18 bacterial suspension and sterile filtrate on microcystis aeruginosa, respectively;
in the figure, A is morphological characteristics of actinomycetes D18 strain;
B is the algae dissolving effect of D18 bacterial liquid, D18 bacterial suspension and sterile filtrate on microcystis aeruginosa respectively, wherein (a) is the D18 bacterial suspension, (B) is the D18 bacterial liquid, and (c) is the sterile filtrate.
FIG. 2 is a graph showing the statistics of the algae dissolution rate of D18 bacterial liquid, D18 bacterial suspension and sterile filtrate on microcystis aeruginosa cells at different culture times.
FIG. 3 shows the effect of light on the algicidal rate of actinomycetes D18.
FIG. 4 is the effect of actinomycetes D18 on the chlorophyll fluorescence parameters of microcystis aeruginosa;
in the graph, A is the influence of sterile filtrate at different culture times on Fv/Fm of microcystis aeruginosa;
b is the influence of the sterile filtrate on microcystis aeruginosa YII in different culture times.
FIG. 5 is the effect of actinomycetes D18 on microcystis aeruginosa phycobiliprotein;
In the figure, A is the effect of actinomycetes D18 on allophycocyanin content;
B is the influence of actinomycetes D18 on the phycocyanin content;
c is the effect of actinomycetes D18 on phycoerythrin content.
FIG. 6 shows the effect of actinomycetes D18 on the carotene content of microcystis aeruginosa.
FIG. 7 is a graph showing the effect of actinomycete D18 on the ATP content of microcystis aeruginosa;
in the figure, A is the effect of actinomycetes D18 on the total ATP concentration of microcystis aeruginosa;
b is the effect of actinomycete D18 on the intracellular ATP concentration of microcystis aeruginosa.
FIG. 8 is an effect of actinomycetes D18 on the antioxidant system of microcystis aeruginosa;
in the figure, A is the effect of actinomycetes D18 on the CAT enzyme activity of microcystis aeruginosa;
b is the effect of actinomycete D18 on the activity of microcystis aeruginosa superoxide dismutase (SOD).
FIG. 9 is a graph showing the effect of actinomycetes D18 on malondialdehyde content (MDA) of Microcystis aeruginosa.
FIG. 10 shows the effect of actinomycetes D18 on the Zeta potential and pH of microcystis aeruginosa, and only the results of the treatment experiments.
FIG. 11 is a three-dimensional fluorescence spectrum characterization of actinomycetes D18 algicidal products;
in the figure, (a) fluorescence intensity analysis of the sterile filtrate prepared for actinomycetes D18;
(b) Analyzing the fluorescence intensity of microcystis aeruginosa liquid;
(c) Analyzing the fluorescence intensity of an initial solution of the mixed solution of the sterile filtrate and the microcystis aeruginosa liquid;
(d) Fluorescence intensity analysis was performed after 10 days of co-culture of the sterile filtrate with microcystis aeruginosa liquid.
FIG. 12 is a denitrification property study of actinomycetes D18;
in the figure, A is the total nitrogen removal efficiency of actinomycetes D18;
B is the removal efficiency of actinomycetes D18 on nitrate (NO 3 - -N);
C is the effect of actinomycetes D18 on nitrite (NO 2 - -N) and ammonia concentration (NH 4 + -N).
FIG. 13 shows denitrification and algicidal effects of actinomycetes D18 in water environmental pollution;
A is the algae dissolving efficiency (algae killing rate) of actinomycetes D18 in raw water;
B is the removal efficiency of actinomycetes D18 on nitrate (NO 3 - -N) in raw water;
C is the influence of actinomycetes D18 on nitrite (NO 2 - -N) and ammonia concentration (NH 4 + -N) in raw water;
d is the removal efficiency of actinomycetes D18 on total nitrogen in raw water.
Detailed Description
The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified, and materials, reagents, etc. used in the examples described below are commercially available.
Example 1: isolation and identification of strain D18 with algicidal function.
1. Experimental method
1. Sampling
Collecting a water source reservoir surface water body: gold basin reservoir (34 degree 32'38 ' N,107 degree 53' E)
2. Isolation and identification of Strain D18
(1) Separating: the water source reservoir is collected and covered with water sample, the water sample is collected in a sterile polyethylene bottle and transferred to a laboratory. For enrichment of actinomycetes, 100mL of the overlaid water sample was filtered through a 0.22 μm polycarbonate membrane, and the filtered polycarbonate membrane was spread on sterilized solid medium of Gao's first order. Since actinomycetes can grow on the solid medium of high-k No. 1 by penetrating the membrane pores, the microorganism-enriched membrane is face-up attached to the solid medium, followed by culturing at 30 ℃ for 6 to 9 days. After one cycle of culture, colonies of different shapes and sizes were formed on solid medium of Gao's No. 1. Due to competing growth of bacteria and fungi, 1-2 plate streaks and pure culture are required for selective isolation of actinomycete colonies. All plate samples were incubated for 6-9 days (3 replicates were set) and then subjected to 1-2 selective separations to form different colonies with specific characteristics of actinomycetes on solid medium at No. one of Gao. To further isolate individual actinomycetes, colonies of different shapes were streaked onto solid plates every 7 days and the process repeated 5-6 times. The purified actinomycete colony has the same shape on a solid plate of Gao's No. 1. Solid plates with the same colony morphology were then selected for further investigation. The isolated strain was designated as D18.
(2) And (3) identification: the isolated strain D18 was subjected to 16S rDNA gene sequence analysis.
3. Determination of algicidal Rate of Strain D18
Determination of the algicidal action mode of strain D18:
(1) D18 bacterial liquid preparation: inoculating actinomycetes D18 to sterilized DM culture medium, and culturing in biochemical shaking incubator at 30deg.C for 130 rpm hr to logarithmic phase to obtain D18 bacterial liquid.
(2) Determination of algae dissolution rate: and (3) centrifuging the D18 bacterial liquid at 10000 r/min and 4 ℃ for 10min under the aseptic condition to obtain a supernatant and thalli. Wherein the supernatant was filtered with a sterile filter membrane of 0.22 μm to obtain a sterile filtrate. The bacterial cells are washed three times by a sterilized BG-11 culture medium and centrifuged, and finally the bacterial cell sediment is resuspended by an equal volume of BG-11 culture medium to obtain the D18 bacterial suspension.
Inoculating the D18 bacterial liquid, the D18 bacterial suspension and the sterile filtrate into the microcystis aeruginosa liquid according to the inoculum size of 20 percent of volume fraction (OD 680 = 0.3); the sterilized DM medium was added to the microcystis aeruginosa solution at the same inoculum size as a control group. Three replicates were set for each treatment, and samples were taken as samples after 10 days of incubation to determine chlorophyll a (Chl-a) and its algicidal rate.
Preparing microcystis aeruginosa liquid: inoculating the purchased M.aerosea FACHB-905 into BG11 culture medium (pH 7.5) according to the volume fraction of 20%, culturing under the condition of illumination 2400 lux for 12h and 12h of light-dark circulation, and culturing until the logarithmic growth phase OD 680 =0.3 to obtain the microcystis aeruginosa liquid.
The M. aeuginosa FACHB-905 is purchased from the institute of Others, china academy of sciences, namely, microcystis aeruginosa FACHB-912 (Microcystis aeruginosa FACHB-912) of the phylum Cyanophyta.
In the period of expanding culture of algae liquid, in order to avoid the problem of uneven illumination, the algae liquid is periodically shaken for three times every day to ensure the normal growth of algae.
The formula of the BG-11 medium, the DM medium and the culture medium of Gaoshi No. one is as follows:
Table 1 BG-11 Medium formulations
A5 (microelement solution) preparation ：H3BO3：2.86 g/LdH2O; MnCl2·4H2O：1.86 g/LdH2O; ZnSO4·7H2O：0.22 g/LdH2O; Na2MoO4·2H2O; CuSO4·5H2O：0.08 g/LdH2O; Co (NO3)2：0.05 g/LdH2O.
TABLE 2 composition of culture medium of Gao's No. 1
TABLE 3 DM composition of culture medium
The chlorophyll a (Chl-a) was measured as follows: 10 The mL samples were centrifuged at 10000 r/min at 4℃for 10: 10min, the supernatant was discarded, and 75% absolute ethanol 10: 10mL was then added. After being wrapped with tinfoil paper for shading treatment, the centrifugal tube is subjected to ultrasonic treatment in a numerical control ultrasonic cleaner with the power of 100Hz for 30 min. After the ultrasonic treatment, the extracted sample is kept stand and extracted for 24h at 4 ℃ in dark. Finally, centrifuging the sample at 10000 r/min and 4 ℃ for 10min times, repeating for 2 times, taking supernatant, measuring absorbance at 630nm, 645nm, 663nm and 750nm by using an ultraviolet spectrophotometer (UV-2600, UNICO, USA), and calculating chlorophyll a concentration by the formula (1):
（1）
Wherein c is chlorophyll a content in μg/L; v 1 is sample volume, 10: 10 mL; v is the liquid volume of microcystis aeruginosa in unit L; delta is the cuvette optical path, 1cm was taken.
The algae dissolution rate is calculated by the formula (2):
Algae dissolution rate (ALGICIDAL RATIO (%)) = [ (C 0 - Ct）/ C0 ] ×100% (2)
Wherein, C 0 is chlorophyll a content of blank control group; c t is chlorophyll a content of the treated group.
2. Experimental results
1. Isolation and identification of Strain D18
The colonies of the isolated strain D18 exhibited a milky yellow, divergent, dry and rough surface (see A of FIG. 1). 16S rDNA identified strain D18 as Streptomyces (Streptomyces venezuelae). And strain D18 was deposited with the chinese collection at 2023, 8 and 10, accession number: cctccc NO: m20231417, classified and named: streptomyces venezuelae D18 and 18.
2. Determination of algicidal Rate of Strain D18
The B in FIG. 1 is the algicidal effect of the D18 bacterial liquid (B), the D18 bacterial suspension (a) and the sterile filtrate (c) on microcystis aeruginosa, respectively, and it can be seen from the figure that the D18 bacterial suspension has no algicidal effect, and the D18 bacterial liquid and the sterile filtrate have obvious algicidal effect.
FIG. 2 shows the algicidal effect of D18 bacterial liquid, D18 bacterial suspension and sterile filtrate on microcystis aeruginosa cells at various treatment times. As seen from the figure, the algae dissolution rates of the D18 bacterial liquid and the sterile filtrate are almost synchronously increased, and reach 85.0% and 83.6% respectively after the 10 th day of culture. And the D18 bacterial suspension and the algae liquid show good growth characteristics after co-culture. This suggests that the algicidal action of actinomycetes D18 is indirect, i.e., algicidal by secretion of extracellular active substances.
Example 2: effect of light on algicidal effect of actinomycetes D18.
1. Experimental method
In this example, the sterile filtrate was co-cultured with M.aerosea FACHB-905 algae (microcystis aeruginosa) in treatment groups and incubated under total light (24 h), total darkness (24 h) and light dark cycle (12:12 h) respectively. The algae solution was shaken periodically daily during the cultivation, sampled and algae dissolution rates were calculated on day 10, respectively.
Treatment group: inoculating the sterile filtrate into microcystis aeruginosa liquid (OD 680 = 0.3) according to the inoculum size of 20% of the volume fraction;
control group: and adding the sterilized DM culture medium to the microcystis aeruginosa liquid according to the same inoculation amount.
The algae dissolution rate calculation method is the same as in example 1.
2. Experimental results
The results are shown in fig. 3, and the algicidal effect of the sterile filtrate is from high to low: light-dark cycle (12:12 h) > full light (24 h) > full darkness (24 h). And the algicidal activity of the sterile filtrate on microcystis aeruginosa is increased by 22.2 percent and 49.3 percent under the condition of light and dark cycle (12:12 h) respectively compared with that under the conditions of full darkness (24 h) and full illumination (24 h). And under the condition of light and dark cycle (12:12 h), the highest algae dissolution rate is 82.24 percent. This suggests that the algicidal activity of D18 is light dependent and that the photosynthetic system of Microcystis aeruginosa is one of the major targets for the action of strain D18. Under the condition of complete darkness, the algae cells can not normally carry out the normal physiological metabolism of the photosynthesis supporting cells, and the damage caused by the normal physiological metabolism can not be repaired in time.
Example 3: effect of actinomycetes D18 on chlorophyll fluorescence parameters of microcystis aeruginosa.
1. Experimental method
In this example, the sterile filtrate was co-cultured with M.aerosea FACHB-905 algae (microcystis aeruginosa) solution (treatment group) and after co-culturing days 0, 2, 4, 6, 8 and 10, the photosynthetic response of algae cells to D18 sterile filtrate was determined using a modulated chlorophyll fluorescence imaging system. Chlorophyll fluorescence parameters include Fv/Fm (maximum quantum yield of photosystem PSII), YII (effective quantum yield of photosystem PSII). 1.5 mL co-cultured mixtures (treatment or control) were placed in centrifuge tubes before each assay and immediately after dark reaction 10 min.
Treatment group: inoculating the sterile filtrate into microcystis aeruginosa liquid according to the inoculation amount of 20% of the volume fraction;
Control group: the sterilized DM culture medium is added into the microcystis aeruginosa liquid according to the same inoculation amount for co-culture.
2. Experimental results
The results are shown in FIG. 4, and the effects of the sterile filtrate on the photosynthetic system of algal cells were evaluated using Fv/Fm and YII. After 10 days of treatment, the Fv/Fm of the control group decreased slightly and then increased gradually, reaching 0.286 after day 10. The Fv/Fm of the treated group decreased with the increase in the culture time, and was shown to be 0 after day 10. YII also showed the same trend, with treatment group values of almost 0 at day 10. After addition of the sterile filtrate, the photosynthetic capacity of the microcystis aeruginosa cells is reduced. This result provides evidence for PSII dysfunction, apparently due to the inhibition of electron flow in PSII. While photosynthesis disorders can result in excessive ROS production by algal cells, excessive ROS can lead to more serious cell damage or cell death.
Example 4: effects of actinomycetes D18 on Microcystis aeruginosa phycobiliprotein.
1. Experimental method
The determination method of phycobiliprotein content comprises the following steps: 5mL of algae solution (treatment group or control group) was centrifuged at 10000 rpm at 4℃for 10min, and then the supernatant was discarded, 5mL of pre-chilled Phosphate Buffer (PBS) with pH=7.8 and 0.05mol/L was added, and the pellet was re-suspended and centrifuged again and washed twice repeatedly. The obtained precipitate sample was added with 5mL precooled PBS with pH=7.8 and 0.05mol/L, and then frozen in a refrigerator for 8 h, dissolved at room temperature, and repeatedly frozen and thawed for 3 times. Wherein dissolution at room temperature requires careful light-shielding operation. The frozen and dissolved sample was centrifuged at 10000 r/min at 4℃for 10: 10min to obtain a supernatant, and the absorbance of the supernatant at 560 nm,620nm and 650nm was measured.
Treatment group: inoculating the sterile filtrate into microcystis aeruginosa liquid according to the inoculation amount of 20% of volume fraction;
Control group: the sterilized DM medium was added to the microcystis aeruginosa solution at the same inoculum size.
Phycobiliproteins include phycocyanin, allophycocyanin and phycoerythrin.
Phycocyanin (PC) is calculated by formula (3):
PC =（OD620-0.7 × OD650）/ 7.38 （3）；
allophycocyanin (APC) is calculated from formula (4):
APC =（OD650-0.19 × OD620）/ 5.65 （4）；
phycoerythrin (PE) is calculated from equation (5):
PE =（OD565-2.8 × PC-1.34 × APC）/ 1.27 （5）。
2. Experimental results
FIG. 5 shows that allophycocyanin content in microcystis aeruginosa liquid without addition of sterile filtrate (control group) increases with prolonged culture time, while allophycocyanin content decreases with prolonged culture time when the sterile filtrate is added to algal cells (treatment group) subjected to exogenous stress. Similarly, phycocyanin and phycoerythrin also show the same trend. This indicates that microcystis aeruginosa is adversely affected by D18 supernatant, and the ability of microcystis aeruginosa to produce three proteins is damaged, the light capturing-transmitting efficiency is weakened, and the pigment molecule content is reduced to inhibit photosynthesis.
Example 5: effect of actinomycetes D18 on the carotene content of microcystis aeruginosa.
1. Experimental method
Taking the algae liquid 10 mL of the control group and the treated group respectively, centrifuging at 10000 rpm and 4 ℃ for 10min, discarding supernatant, adding 75% absolute ethanol 10 mL, and re-suspending and uniformly mixing. And then carrying out ultrasonic crushing treatment on the centrifuge tube in a numerical control ultrasonic cleaner with the power of 100 Hz after shading the centrifuge tube for 30 min. After the ultrasonic treatment is finished, the taken sample is subjected to light-shielding static extraction at 4 ℃ for 24 h. Next, the sample was centrifuged at 10000 rpm at 4℃for 10min times, and the supernatant was collected and absorbance of the sample at 480nm, 630nm and 664nm was measured.
Treatment group: inoculating the sterile filtrate into microcystis aeruginosa liquid according to the proportion of 20% (v/v);
control group: and adding the sterilized DM culture medium to the microcystis aeruginosa liquid according to the same inoculation amount.
2. Experimental results
As a result, as shown in FIG. 6, carotenoids in the treated group to which the sterile filtrate was added gradually decreased with the lapse of the culture time, by 0.116 mg/L after the 10 th day, as compared with the control group. The control group increased steadily with time, and after 10 days of culture, the carotenoid content of the control group was 3.0 times that of the treatment group. This suggests that the sterile filtrate produced by D18 may cause oxidative stress in the algal cells. Along with the prolongation of the stress time, the microcystis aeruginosa cell function metabolism is possibly damaged, and photosynthesis is destroyed, so that the capturing can not be performed efficiently, and the synthesis of carotenoid is indirectly influenced. The addition of the sterile filtrate affects the conversion of carotenoids, and the decrease in pigment content causes a decrease in the fluorescence parameters of the chlorophyll of the photosystem. The decrease in photosynthetic capacity indicates that microcystis aeruginosa cells have reduced autotrophic capacity and limited cell growth.
Example 6: effect of actinomycetes D18 on ATP content of microcystis aeruginosa.
1. Experimental method
Total ATP and extracellular ATP content were measured using a biochemistry luminometer (Promega, WI, USA) to assess algae viability. The ATP staining agent is prepared by mixing substrate freeze-dried powder and buffer solution in Bac-Titer-Glo ™ (G8231, promega, USA) kit, wrapping with tinfoil paper, and storing in a refrigerator at-20deg.C. For the measurement, 500. Mu.L of the sample and 50. Mu.LATP stain were first placed in a 1.5mL centrifuge tube and heated in a metal bath (DHB-smart, DHS, china) at a temperature set to 38℃for 10 min and 2 min, respectively. After heating the sample and ATP stain, the two are mixed well and heated to 20 s f. Immediately after the heating was completed, the luminous intensity was measured. The sample was filtered through a 0.1 μm sterile filter to obtain an extracellular ATP sample. Extracellular ATP samples were assayed as described above. Finally, the intracellular ATP obtained is the total ATP minus extracellular ATP.
Samples included treatment and control samples, treatment: inoculating the sterile filtrate into microcystis aeruginosa liquid according to the inoculation amount of 20% of volume fraction; control group: and adding the sterilized DM culture medium to the microcystis aeruginosa liquid according to the same inoculation amount.
2. Experimental results
As shown in FIG. 7, the total ATP content of the control cells steadily increased with the increase of the culture time, which indicates that the Microcystis aeruginosa cells are in a normal growth state. In contrast, after adding the sterile filtrate to the algae solution (treatment group) for co-cultivation, the total ATP content of the treatment group started to decrease, which was almost 0 after detection on days 8 and 10. Similar changes in intracellular ATP content occur. This suggests that under the stress of certain chemicals in the sterile filtrate, an imbalance in ATP production and consumption results in abnormal growth of microcystis aeruginosa cells.
Treatment group: inoculating the sterile filtrate into microcystis aeruginosa liquid (OD 680 =0.3) according to the inoculum size of 20% of the volume fraction;
control group: and adding the sterilized DM culture medium to the microcystis aeruginosa liquid according to the same inoculation amount.
Example 7: influence of actinomycetes D18 on the antioxidant system of Microcystis aeruginosa.
1. Test method
Superoxide dismutase (SOD) activity, CAT enzyme activity and malondialdehyde content (MDA) were measured in the treated and control groups as follows.
Treatment group: inoculating the sterile filtrate into microcystis aeruginosa liquid according to the inoculum size of 20% of the volume fraction for co-cultivation;
control group: and adding the sterilized DM culture medium to the microcystis aeruginosa liquid according to the same inoculation amount.
The preparation method of the crude enzyme liquid comprises the following steps: shaking the algae liquid in the treatment group uniformly, accurately taking 10mL in a sterile centrifuge tube, centrifuging at 10000 rpm and 4 ℃ for 10min, and discarding the supernatant to obtain cell sediment. Adding 5mL pre-cooled phosphate buffer solution into the obtained precipitate, shaking, centrifuging again at 10000 rpm and 4deg.C for 10min times, and repeating for 2-3 times to obtain algae cell precipitate. Then, after adding 4 mL and 0.05 mol/L PBS (ph=7.8) to the algal cell pellet, disruption was performed using an ultrasonic cytodisruption instrument. The ice bath is carried out during crushing, the parameters are set to be ultrasonic 3s, interval 5 s, working time 4 min, power 150W and repeated three times. After crushing, centrifugal 10min is carried out again at 10000 rpm and 4 ℃, and supernatant fluid is collected, namely crude enzyme liquid. The crude enzyme solution was carefully aspirated and stored in a refrigerator at 4℃for subsequent enzyme activity and malondialdehyde content (MDA) determination.
The method for measuring the protein content adopts a Coomassie brilliant blue G-250 and proteome combination method. First, the protein standard curve was measured: 0.1 mg/L standard protein solution is prepared by using bovine serum albumin, and 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mL are respectively added into 7 test tubes, and then the volume is fixed to 1 mL by using ultrapure water. Next, 5 mL Coomassie brilliant blue G-250 solution was added in one portion to the 7 tubes. Immediately after mixing, the mixture was left to develop color 5min, and the absorbance (OD 595) of each tube was measured by adjusting the wavelength to 595 nm. Protein content standard curves are obtained by taking standard protein concentration mg/L as an abscissa and OD 595 as an ordinate: y=0.0057 x + 0.0839 (R 2 =0.997). The protein content of the sample can be calculated by substituting the measured OD 595 value into a standard curve.
The measurement method of the superoxide dismutase (SOD) activity is as follows: the absorbance of the sample at 450nm was measured according to the instruction of the SOD superoxide dismutase activity detection kit (LABLEAD, no. S0311), then the inhibition rate was calculated according to the instruction calculation formula, and then the SOD enzyme activity was determined and expressed in units of U/mg.
The CAT enzyme activity was determined as follows: the CAT activity of cells was determined in this experiment according to the instructions of the Catalase (CAT) activity detection kit (Solarbio, no. BC0250). The prepared crude enzyme solution is measured by using a 96-well plate and an enzyme-labeled instrument. The viability of the cell CAT was then calculated and determined according to the kit instructions.
The method for measuring MDA content is as follows: the thiobarbituric acid-malondialdehyde method (MDA-TBA) was used. Taking the prepared crude enzyme solution 2 mL in a colorimetric tube with a plug, adding 2 mL ultrapure water (manufacturer: millipore, USA) into a blank control tube, adding 2 mL of 0.6% of the existing thiobarbituric acid solution, and shaking thoroughly. The sample was placed in a boiling water bath for reaction 15 min, at which time the solution in the tube appeared to be small bubbles. After removal, the sample was cooled rapidly and centrifuged at 10000 rpm and 4℃for 10 min to obtain a supernatant. After calibration with a blank control tube, the absorbance of the supernatant samples was measured at 532nm, 600nm and 450 nm. The MDA content is calculated by the formula (6).
MDA content (μmol/L) =6.45 (OD 532-OD600）-0.56OD450 (6).
2. Experimental results
The results are shown in FIG. 8, in which CAT and SOD activities of the control group remained substantially stable after 10 days of culture. Whereas the enzyme activity of the treated group increased first, reached a peak at day 6, and then the enzyme activity began to decrease, but was consistently higher than the control group. Therefore, after the microcystis aeruginosa cells are stressed by sterile filtrate, an antioxidant system is activated, and higher enzyme activity is maintained. And under continuous exposure, the antioxidant enzyme activity starts to decline, which indicates that the aseptic filtrate causes the oxidative stress of microcystis aeruginosa cells and simultaneously interferes with the enzymatic antioxidant system of the microcystis aeruginosa cells. Antioxidant systems are not resistant to sustained damage and accumulation of peroxidation products (e.g., MDA). These conditions trigger structural and functional destruction of microcystis aeruginosa cells, ultimately leading to dysfunction of the membrane system, leading to microcystis aeruginosa cell death. In addition, excessive ROS formation may also be due to the extracellular active species generated by D18 inhibiting electron transfer in the electron transfer chain in thylakoid membranes.
As shown in FIG. 9, the MDA content of the treatment group increased to 0.156. Mu. Mol/L on day 2 and reached a peak of 0.281. Mu. Mol/L on day 4, indicating a high MDA level. MDA was in a decreasing trend from day 6 of exposure to the end of the experiment. This suggests that ROS cannot be cleared in time, destroying the cell membrane structure of algae, and impairing the physiological activities of cells, thereby accelerating cell death.
Example 8: effects of actinomycetes D18 on the Zeta potential and pH of Microcystis aeruginosa.
1. Experimental method
Algae solutions of 3mL control and treatment groups were taken in sterile centrifuge tubes on days 0, 2, 4, 6, 8 and 10 of co-cultivation, respectively, and the Zeta potential of the algae cell surface was measured using a Nano ZS90 malvern Zeta potentiometer. The pH was measured using a pH meter (Hach, loveland, CO, USA). Each sample was measured three times and the average Zeta potential value and pH value were determined.
Treatment group: inoculating the sterile filtrate into microcystis aeruginosa liquid according to the inoculum size of 20% of the volume fraction for co-culture;
control group: and adding the sterilized DM culture medium to the microcystis aeruginosa liquid according to the same inoculation amount.
2. Experimental results
As a result, as shown in FIG. 10, after 10 days of co-culture of the treated group, the Zeta potential of the microcystis aeruginosa cell surface was changed from the initial value of-37.95 mV to-15.78 mV. The Zeta potential of the microcystis aeruginosa cells of the control group is generally negative. In the treatment process, the Zeta potential of the microcystis aeruginosa cells in the treatment group is continuously increased, namely the Zeta potential absolute value is continuously reduced. The absolute value of Zeta potential reflects the stability of algal culture. The Zeta value is very low and the algae cells will aggregate.
FIG. 10 shows that when cells die after 10 days, the pH of the co-culture broth (algae broth) of the treatment group increased to 8.88. It is possible that the positive charge of the supernatant neutralizes the negative charge on the surface of the algal cells, raising the pH and Zeta potential. As the treatment time increases, the algae cells die, the algae culture cannot return to a steady state, and the pH is continuously raised by alkaline substances produced by algae metabolism.
Example 9: and (5) performing three-dimensional fluorescence spectrum characteristic analysis on the actinomycete D18 algicidal product.
1. Test method
And (3) adopting three-dimensional fluorescence spectrum analysis to characterize the strain algae-dissolving product, and simultaneously, examining the fluorescence spectrum characteristics of the pure algae liquid and the sterile filtrate. The sample was measured by an instrument F-7000 fluorescence spectrophotometer (Japan). Wherein the instrument parameters are set as: emission wavelength (Em): 250-550nm, excitation wavelength (Ex): 200-450 pmt voltage: 600 V, scan speed: 12000 nm/min, slit width: 5 nm, scan interval: 5 nm. First microcystis aeruginosa was grown to logarithmic phase as algae liquid (b), initial OD 680 =0.3. The sterile filtrate (a) was consistent with the procedure of example 1. Next, the mixed solution (c) of the co-cultured algae solution (treatment group) at the initial day 0 and the degradation product (d) after the end day 10 were measured. The four different samples were measured after filtration through a 0.45 μm sterile filter. Wherein the ultra-pure water is used as blank to correct Raman scattering and Rayleigh scattering.
Treatment group: inoculating the sterile filtrate into microcystis aeruginosa liquid according to the inoculation amount of 20% of volume fraction for co-culture (OD 680 = 0.3);
control group: and adding the sterilized DM culture medium to the microcystis aeruginosa liquid according to the same inoculation amount.
2. Experimental results
As shown in FIG. 11, it was found that some components of humic acid substances were reduced during the algicidal process. Meanwhile, the fluorescence intensity of the aromatic protein and the soluble microorganism metabolite can be increased, which indicates that the addition of the sterile filtrate causes massive rupture and death of microcystis aeruginosa cells, and the intracellular substances are released, so that stronger fluorescence intensity is detected.
Example 10: denitrification property study of actinomycetes D18.
1. Experimental method
Actinomycetes D18 is inoculated into a sterilized DM culture medium, cultured for 48 hours to logarithmic growth phase in a biochemical oscillation incubator at 30 ℃,130 rpm percent, then inoculated into microcystis aeruginosa liquid according to the inoculation amount of 20 percent of volume fraction to be used as a treatment group, and meanwhile, the sterilized DM culture medium is added into the same volume of algae liquid according to the same inoculation amount to be used as a control group. The experimental group and the control group are all provided with three groups in parallel. The culture solutions of the treatment group and the control group are respectively and evenly shaken and then put into an illumination incubator for culture, the temperature is set to 28 ℃, the illumination intensity is 2400 lux, and the light and dark cycle is 12:12 h. The samples were filtered through a pre-burning 0.45 μm GF/F glass fiber filter every 2 days and the concentrations of NO 3 --N、NH4 +-N、NO2 - -N and TN were determined.
2. Experimental results
As a result, actinomycetes D18 exhibited good denitrification properties, as shown in FIG. 12. A significant trend in decrease occurred with prolonged incubation time in the treatment group. After 10 days of reaction, the TN concentration is reduced from the original 231.86 mg/L to 27.57 mg/L, and the TN removal rate can reach 88.1 percent. However, the TN concentration in the control group was somewhat reduced, but remained relatively stable, with TN removal rates at day 10 being only 0.18 times that in the treated group. The NO 3 - -N concentration at day 2, 203.14 mg/L, decreased to day 8, 20.59 mg/L, indicating that the denitrification capacity was very strong and then smoothed. The removal rate of NO 3 - -N in the whole process can reach 96.05 percent. Accumulation of NO 2 - -N occurred, reaching peak 47.76 mg/L on day 6, after which it began to drop to nearly the initial value. The ammonia nitrogen does not change much in the whole process. However, at the end of the reaction time, the NH 4 + -N increase may be due to the decreasing nitrogen source and the strain begins to die. By combining the results of the previous studies, we found that strain D18 itself can be cultured to denitrify denitrification and also produce algicidal substances to kill algae. Actinomycetes D18 can be applied to eutrophic water, and can control and regulate abnormal proliferation of algae and nitrogen pollution of the water.
Example 11: the actinomycetes D18 has the effects of denitrification and algae dissolution in water environment pollution.
1. Experimental method
The Xian city park (CL) is selected as a research point for collecting water samples. Water sample collection at month 8 of 2022, water sample 25: 25L on the surface (0.5-1.0 m) was collected using a sterile polypropylene container. All samples were placed in a cooler and transferred to a laboratory for laboratory simulation experiments to investigate the effect of strain D18 on raw water algae control. Wherein the water quality parameter is ：pH：7.56 ± 0.20;WT：26.1 ± 0.3℃;TN：4.25 ± 0.07 mg/L;NO3 --N：3.29 ± 0.00 mg/L;NO2 --N： 0.04 ± 0.01 mg/L;NH4 +-N：0.53 ± 0.00 mg/L;TP：0.22 ± 0.01 mg/L;CODMn：5.22 ± 0.26 mg/L;Fe：0.03 ± 0.00 mg/L;Mn：0.01 ± 0.00 mg/L.
The reaction was carried out using a 5L glass reactor. Before the experiment starts, the sterile sponge is filled into plastic balls and is taken as a carrier to be put into a 2L plastic reactor, 1.6L of DM culture medium and 0.3L of actinomycete D18 bacteria solution are added, and the mixture is cultivated under aerobic condition (DO=6.0 mg/L) until obvious yellow biological film is formed. Then, the spheres successfully coated were placed in a glass reactor 5L containing 4.5L lake raw water as experimental group (D18-T) and run at room temperature under aerobic conditions for 10 days. In addition, the influence of indigenous bacterial communities in raw water is avoided, and a raw water reactor (C) is also established. Samples were taken on days 0, 2,4, 6, 8, 10, respectively. Chl-a, TN, NO 3 --N, NO2 --N, NH4 + -N concentrations were determined (3 replicates were set).
2. Experimental results
As a result, as shown in FIG. 13, the chlorophyll a concentration in the experimental group (D18-treated group) was reduced from the initial 125.73. Mu.g/L to 29.27. Mu.g/L on the 10 th day, which was 4.3 times the initial concentration, and the algae removal rate was 76.7%. In FIG. 13A, chlorophyll a of the control group was lowered to 904.27. Mu.g/L (90.427 mg/L).
The changes in TN, NO 3 - -N concentration, removal rate and NH 4 +-N、NO2 - -N concentration with time of incubation are shown in FIG. 13. Strain D18 exhibited good denitrification performance. The TN concentration of the experimental group generally showed a decreasing trend from 4.25 mg/L to 0.89mg/L, and the removal rate reached 79.16% (D of FIG. 13), indicating that the D18 strain had good denitrification performance. An increase in total nitrogen concentration was observed on day 2. The same phenomenon was also observed with B of FIG. 13, with NO 3 - -N concentration decreasing from the initial 3.29: 3.29 mg/L to 0.61: 0.61 mg/L on day 10, with removal of 81.4%. The treatment group showed significant accumulation of NO 2 - -N compared to the control group, but was again reduced to 0.20 mg/L after the end of the experiment. The accumulation of nitrite nitrogen may be due to a continuous lag in the reduction of nitrate and nitrite.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (7)
1. Actinomycetes (Streptomyces venezuelae) D18 with algae dissolving function, wherein actinomycetes D18 is preserved in China center for type culture Collection, with a preservation number of 2023, 8 and 10: cctccc NO: m20231417.
2. A bacterial liquid prepared from actinomycetes D18 according to claim 1.
3. A method for preparing a bacterial liquid according to claim 2, wherein the D18 bacterial liquid is obtained by inoculating the D18 bacterial strain into a DM culture medium and culturing the culture medium at 28-30 ℃ and 130-150rpm until the culture medium is in a logarithmic growth phase.
4. A sterile filtrate prepared from actinomycetes D18 according to claim 1.
5. Use of actinomycetes D18 according to claim 1, bacterial liquid according to claim 2 or sterile filtrate according to claim 4 in water treatment, characterized in that actinomycetes D18, bacterial liquid or sterile filtrate is used for removal of microcystis aeruginosa in an aqueous environment.
6. The use according to claim 5, characterized in that actinomycetes D18, bacterial liquid or sterile filtrate is used for denitrification of sewage or wastewater.
7. The use according to claim 6, wherein the denitrification is to reduce the total nitrogen, nitrate, nitrite and ammonia content in the aqueous environment.
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