CN113755336B - Chlorella strain resistant to pollution and application thereof in livestock and poultry waste treatment - Google Patents

Chlorella strain resistant to pollution and application thereof in livestock and poultry waste treatment Download PDF

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CN113755336B
CN113755336B CN202111130444.3A CN202111130444A CN113755336B CN 113755336 B CN113755336 B CN 113755336B CN 202111130444 A CN202111130444 A CN 202111130444A CN 113755336 B CN113755336 B CN 113755336B
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邓兵
孔丹妮
高其双
濮振宇
彭霞
邵中保
秦心儿
冉志平
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F2305/06—Nutrients for stimulating the growth of microorganisms

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Abstract

The invention discloses a chlorella DTG5 which is preserved in China center for type culture collection (CCTCC NO) with the preservation number of CCTCCNO: m2021297; the algae strain is separated from the cultivation wastewater, can assimilate and absorb nutrient salts such as nitrogen and phosphorus under photosynthesis, realizes high-efficiency denitrification and dephosphorization, has good growth in pig farm biogas slurry, has strong removal capability for ammonia nitrogen and total phosphorus in the biogas slurry, has removal rates respectively reaching 91.93 percent and 98.14 percent after 8 days of inoculation, is suitable for purifying pig farm biogas slurry, and realizes resource utilization.

Description

Chlorella strain resistant to pollution and application thereof in livestock and poultry waste treatment
Technical Field
The invention belongs to the field of waste treatment in livestock breeding, and particularly relates to a strain of pollution-resistant chlorella and application thereof in livestock waste treatment.
Background
With the rapid development of Chinese economy, the living standard of people is gradually improved, the demands for livestock products are increased, and the livestock breeding products produced by the traditional breeding mode are insufficient to meet the market demands, so that the livestock breeding industry of China is continuously developed towards the large-scale and intensive direction. Livestock and poultry waste in a farm is usually treated by utilizing a biogas project, clean energy biogas can be generated while livestock and poultry waste is treated, the recycling of the waste can be realized, and the concept of sustainable development of human society is met. However, with the popularization and utilization of biogas engineering, large and medium-sized farms can generate a large amount of concentrated biogas slurry, the concentration of the biogas slurry is usually high, and the biogas slurry is rich in a large amount of nutrient substances such as nitrogen, phosphorus, potassium and the like, and if the biogas slurry is directly discharged without treatment, secondary pollution to the environment is easily caused. At present, the biogas slurry treatment in China has a plurality of problems, such as insufficient land consumption, excessive investment of biochemical treatment equipment, poor effect and the like.
The biogas slurry is liquid produced by anaerobic fermentation of livestock and poultry waste, contains rich amino acids, auxin, mineral elements, indoleacetic acid, lactobacillus, bacillus, gibberellin and other elements besides a large amount of nutrient elements such as nitrogen, phosphorus, potassium and the like and trace elements such as zinc, calcium, iron, manganese and the like. The nutrient elements basically exist in the form of available nutrients, so that the biogas slurry has strong available nutrient capability and high nutrient availability, is a multi-element available compound fertilizer, and can be rapidly absorbed and utilized by animals and crops. Therefore, although the biogas slurry is a waste water, if the biogas slurry can be recycled by a technology, the biogas slurry can be used as a resource with rich nutrient components.
Microalgae is a low-grade single-cell photosynthetic organism, has strong photosynthetic capacity, high growth speed and short growth period, is a main advantage of the microalgae, and can absorb a large amount of N, P and other nutritive salt substances in water in a short period. Microalgae have low requirements on growth environment, have extremely strong adaptability, are easy to culture, and can grow in ponds, rivers, deserts, gobi and the like. Microalgae are various, the physiological and biochemical characteristics of the microalgae are widely applied, and in the beginning of the 21 st century, more than thirty thousand algae are found, wherein about 70% of the microalgae account for, and different types of microalgae contain a plurality of bioactive substances with unique functions, such as fat, protein and the like. The culture medium for artificially culturing microalgae needs to contain enough elements such as carbon, nitrogen, phosphorus and the like, and the elements exist in a large amount in the culture biogas slurry, so that the treatment and purification of the biogas slurry by utilizing the microalgae quickly becomes a research hot spot in the field of livestock and poultry environment bioengineering. The microalgae can assimilate and absorb nutrient salts such as nitrogen and phosphorus in the biogas slurry under photosynthesis, so that the efficient denitrification and dephosphorization effects are realized, and the biogas slurry reaches the emission standard.
The microalgae are used for treating the biogas slurry, but the technical difficulty also exists, the tolerance degree of different microalgae to the biogas slurry is different, and the purifying effect is different. Finding a microalgae strain with good tolerance to biogas slurry, the strain can grow well in a certain range of biogas slurry, and can efficiently remove substances such as nitrogen, phosphorus and the like in the biogas slurry.
Disclosure of Invention
The invention aims to overcome the defects of different microalgae in the prior art that the tolerance degree of different microalgae to biogas slurry is different, the purifying effect is different, and microalgae strains with better tolerance are lacking, and provides a strain of pollution-resistant chlorella and application thereof in livestock and poultry waste treatment.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a strain of pollution-resistant chlorella DTG5, which is classified and named as Chlorella vulgaris (Chlorophyta) DTG5, and is deposited in China center for type culture Collection with the deposit number
Cctccc NO: m2021297, the preservation address is university of Wuhan in Wuhan, china, and the preservation date is 2021, 3 and 29 days; the sequence information is shown as SEQ ID NO.1, and the similarity of the sequence of the polypeptide with other sequences is lower than 97.6%.
The invention discloses a chlorella DTG5 which is separated from a multistage discharge pool of culture wastewater in a farm, belongs to the phylum Chlorophyta, the class Chlorophyceae, the order Chlorophyceae, the genus Chlorella, is green, mostly single-cell algae, a few of which are aggregated into multiple cells, the cell morphology is spherical or elliptic, pigment occupies half or more of cells, has cell nuclei and has the diameter of about 3-8 mu m.
The chlorella DTG5 is separated from a water sample in a second treatment tank of a multistage discharge tank of the culture wastewater of a farm, and the separation and purification methods are as follows: and (3) filtering the collected water sample by using a single layer of 200-300 meshes of gauze, taking 200 mu L of filtered water sample into a 96-well plate, gradually diluting the water sample to 8-10 holes, and observing dominant algae species in the water sample under a microscope.
Adding a water sample diluted to a certain degree into a solid culture medium, uniformly scratching the water sample into the solid culture medium by using a coating rod, finding obvious algae fall after 7-10 days, searching pure algae seeds by microscopic examination, and inoculating the pure algae seeds into a 1.5mL centrifuge tube filled with a liquid culture medium by using an inoculating loop. Placing in an incubator for culturing.
The method for measuring the optimal growth concentration of the chlorella DTG5 comprises the following steps: and (3) collecting the original biogas slurry of Dongtai livestock Co, diluting the original biogas slurry with pure water according to the proportion of 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32, and inoculating and screening to obtain the microalgae.
Wherein, the culture conditions of the chlorella DTG5 are as follows: the temperature was 25 ℃ + -1 ℃.
Preferably, when the chlorella DTG5 is cultured in biogas slurry, the illumination intensity is 30-40% (4000 Lux), the light-dark ratio is 12h to 12h, and the chlorella DTG5 is shaken twice a day in the morning and evening.
The relative growth rate of 1-12 d is calculated, and the calculation formula is as follows: k= (InN 2 -InN 1 )/(t 2 -t 1 )
Wherein K is the relative growth rate, t 1 、t 2 The culture time is corresponding to the culture time; n (N) 1 、N 2 Respectively t 1 、t 2 OD of the algae liquid in the period 680 nm value.
The invention protects the application of chlorella DTG5 in livestock and poultry waste treatment, in particular to purification of biogas slurry.
The method for purifying biogas slurry by using the chlorella DTG5 comprises the following steps:
diluting the collected raw biogas slurry with pure water according to the ratio of 1:4 optimal biogas slurry growth concentration, and selecting microalgae (OD) with logarithmic growth phase 680 nm value is about 1.0) is inoculated into diluted biogas slurry according to the ratio of 1:10.
The microalgae has good removal effect on harmful substances such as high-concentration nitrogen, phosphorus and the like in the biogas slurry, but researches find that the biogas slurry directly produced by a culturing farm has high concentration and high turbidity, light is difficult to penetrate, photosynthesis of the microalgae can be seriously affected, growth of the microalgae in the biogas slurry is affected, death of the microalgae can be caused, and the purification effect on the biogas slurry can be reduced. Therefore, a pretreatment method such as dilution can be adopted to reduce the concentration of biogas slurry and the degree of light transmission.
The detection indexes of the biogas slurry purification effect are chemical oxygen demand, total phosphorus, total nitrogen and ammonia nitrogen.
The total nitrogen and total phosphorus removal rate (r) has the following calculation formula:
r＝(C 0 －C t )/C 0 ×100％
wherein C is 0 Is the initial total nitrogen total phosphorus concentration;
C t the concentration (mg/L) after t days of culture was determined.
The invention has the beneficial effects that:
the chlorella DTG5 can grow in the properly diluted biogas slurry of the farm, and takes nutrient substances such as nitrogen, phosphorus, potassium and the like in the biogas slurry as nitrogen sources and carbon sources for self growth, so that the high-efficiency denitrification and dephosphorization effects are achieved on the biogas slurry. The chlorella disclosed by the invention has a good effect of removing ammonia nitrogen and total phosphorus in biogas slurry, and the removal rate in 8 days can reach 98.14% and 91.93% respectively.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 shows the cell morphology of the stain-resistant Chlorella DTG5 strain of the present invention under an optical microscope;
FIG. 2 is a phylogenetic tree constructed based on the pollution-resistant chlorella DTG5 in the invention;
FIG. 3 shows growth curves of the pollution-resistant chlorella DTG5 obtained by separation and purification under different concentrations of biogas slurry;
FIG. 4 is a comparison of the separation and purification of the pollution-resistant chlorella DTG5 and the removal of ammonia nitrogen from biogas slurry by two other strains of algae separated and purified from biogas slurry;
FIG. 5 is a comparison of the total nitrogen removal of the chlorella vulgaris DTG5 with the other two strains separated and purified from the biogas slurry;
FIG. 6 shows the comparison of the total phosphorus removal of the chlorella vulgaris DTG5 with the other two strains separated and purified from the biogas slurry;
FIG. 7 shows a comparison of the DTG5 sequence with other species.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
Example 1
Isolation and purification of algal strains
(1) Sample collection
The algae strain is separated from a water sample collected in a second treatment tank of a multi-stage discharge tank of the culture wastewater of the Dongtai livestock raising company. Water samples were collected from the selected sites, placed in Erlenmeyer flasks and kept in a refrigerator at 4 ℃ for further use.
(2) Isolation, purification and cultivation of algal strains
The microalgae is separated by adopting a method combining a dilution separation method and a solid culture medium. Filtering the collected microalgae liquid with 200-300 mesh gauze, diluting biogas slurry by using a 96-well plate according to a gradient, uniformly coating on a BG-11 solid culture medium by using a coating rod after diluting to a proper degree until colonies on a flat plate are single, observing whether the cultured microalgae strains are consistent in cell morphology by using an optical microscope, if so, achieving the purpose of separation, and if not, repeating the work of coating the flat plate until the cell morphology is single. Inoculating the strain into a 1.5mL centrifuge tube filled with BG-11 liquid culture medium by an inoculating loop, and culturing in an illumination incubator until green algae are behind visible by naked eyes, and shaking the centrifuge tube every day periodically.
Table 1 shows the formulation and amounts of BG-11 medium
BG11 culture medium formula
Figure BDA0003280325440000061
Note that: A5+Co mother liquor is H 3 BO 3 2.86g/l；MnCl 2 ·4H 2 O 1.81g/１；ZnSO 4 ·7H 2 O 0.222g/l；CuSO 4 · 5H 2 O 0.079g/l；NaMoO 4 ·2H 2 O 0.390g/l；Co(NO 3 ) 2 ·6H 2 O 0.0494g/l
Identification and preservation of algal strains
(1) Identification of algal strains
The algae strain is identified by observing its form under an optical microscope, judging by books such as 'Chinese fresh water algae' and the like, and then carrying out molecular biology identification. The characteristics of the strain such as cell morphology, size and structure were observed. The morphology of the algal strain under observation by an optical microscope is shown in FIG. 1.
The pollution-resistant chlorella DTG5 is observed under a microscope, the algae body is green, most of the algae body is single-cell algae, few of the algae body is multicellular aggregation, the cell morphology is spherical or elliptic, the pigment body occupies half or more of cells and has cell nuclei, and the diameter is about 3-8 mu m. Molecular biology identification is carried out on the algae strain, the extracted DNA is taken as a template, a primer is selected from a chlorella general primer, and the primer sequence is as follows: an upstream primer (Forward) RP8 5'-ACC TGG TTG ATC CTG CCA GTA G-3'; downstream primer (Reverse) RP9 5'-ACC TTG TTA CGA CTT CTC CTT CCT C-3'. PCR amplification was performed and a 50. Mu.L PCR reaction system was established. Wherein the amount of the DNA template is 5 to 8. Mu.L, and the amount of each of the upstream and downstream primers is 2. Mu.L, and 2X Taq Plus Master Mix is 25. Mu.L. The PCR amplification reaction conditions were set as follows: pre-deforming at 94 ℃ for 5min, then denaturing at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extending at 72 ℃ for 60s, 30 cycles altogether, and finally extending thoroughly at 72 ℃ for 7min. After the reaction is completed, 5 mu L of PCR products are taken for agarose gel electrophoresis, and then the obtained positive clone products are screened and are entrusted to sequencing by a company.
Comparing the obtained results with NCBI database to obtain sequencing results, downloading similar 18rRNA partial sequences from the database, and carrying out homology analysis on the gene sequences by using BLAST software to find that the sequence has high similarity with chlorella sp and belongs to one branch of chlorella. And (3) carrying out multi-sequence alignment arrangement on the detected sequence and the downloaded sequence by using MEGA4.0 software, constructing a evolutionary tree by adopting a neighbor-joining algorithm, and checking the stability and consistency of the evolutionary tree by using a Bootstrap method, wherein each phylogenetic tree is subjected to 500 self-expanding analyses. The constructed phylogenetic tree of the pollution-resistant chlorella DTG5 is shown in figure 2.
(2) Preservation of algal strains
Inoculating the screened pollution-resistant chlorella DTG5 into a BG-11 culture medium, and placing the culture medium into an incubator for culture under the following conditions: the temperature is 25+/-1 ℃, the illumination is 40% (4000 Lux), the light-dark ratio is 12h to 12h, and the shaking is carried out twice a day in the morning and evening.
The algae strain grows to a certain concentration, and is preserved by a low-temperature preservation method and a secondary preservation method.
The method for preserving the pollution-resistant chlorella DTG5 by using the low temperature comprises the following steps: centrifuging chlorella DTG5 grown to a certain concentration, mixing with frozen stock solution (10% DMSO+90% BG-11 liquid culture medium), standing at 4deg.C for 30min, transferring into a refrigerator at-20deg.C for 30min, and transferring into a liquid nitrogen tank for long-term storage.
The method for applying the secondary preservation method to the pollution-resistant chlorella DTG5 comprises the following steps: the algae strain is inoculated and cultured under proper illumination condition, and when the algae cells grow and propagate to reach higher density and obvious strip or block algae cell groups are seen, the algae cells are transferred to low temperature (4 ℃) and are preserved under the condition of weak light.
The pollution-resistant chlorella DTG5 strain obtained and identified through separation and purification is preserved in China Center for Type Culture Collection (CCTCC) NO: m2021297.
In order to explore the capability of the separated, purified and preserved chlorella strain DTG5 for treating livestock and poultry waste, the method is applied to removal of eutrophic substances in livestock breeding biogas slurry, and the experimental study of chlorella DTG5 cultured in optimal biogas slurry dilution concentration is developed.
In the following experiments, all test groups were set up in triplicate.
Exploration of optimal growth concentration of algae strain
After screening chlorella DTG5 with purification effect, exploring the optimal dilution concentration of the biogas slurry of the microalgae, carrying out gradient dilution on the raw biogas slurry collected from Totai livestock company, inoculating the algae strain, taking a BG-11 culture medium treatment group as a control group, measuring OD680 nm once every two days, and drawing the growth curves of the algae strain of each concentration of biogas slurry and the control group. The growth curve of chlorella DTG5 under different concentrations of biogas slurry is shown in figure 3. Wherein, the biological accumulation amount of the chlorella DTG5 in the 1:4 dilution group in each treatment group is maximum, the chlorella DTG5 is more suitable for the growth of algae strains, the growth trend is basically consistent with that of BG-11 culture medium culture, and the OD680 nm value is higher than that of a control group.
The culture biogas slurry used in the test is taken from stock biogas slurry of Totai livestock Co, the color of the treated biogas slurry is turbid and contains a large amount of impurities, photosynthesis of microalgae is affected and cannot grow normally, the untreated biogas slurry contains substances such as protozoa, bacteria, fungi and the like, especially fungus organisms can grow rapidly in a culture medium, resources are preempted to cause slow growth of the microalgae until the microalgae does not grow, and therefore, the biogas slurry is subjected to treatment such as autoclaving, dilution and the like.
TABLE 2 physicochemical index of biogas slurry before inoculation (Unit: mg/L)
Figure BDA0003280325440000081
The collected raw biogas slurry is subjected to high-pressure sterilization and diluted with pure water according to the proportion of 1:4 to obtain the optimal growth concentration of the biogas slurry, and microalgae (OD) growing to logarithmic phase 680 nm is about 1.0) is inoculated into diluted biogas slurry according to the proportion of 1:10, and the diluted biogas slurry is placed into an incubator with the temperature of 25+/-1 ℃ and the illumination intensity of 40% (4000 Lux) and the light-dark ratio of 12h to 12h for culture.
Comparative example
Gracilaria DTY2 and Chlorella DTG3 screened from the multistage discharge pond II of the culture wastewater of the Dongtai livestock-raising company in the Chlorella DTG5 of the invention are used as comparison, and are cultured to a growth log phase under the same conditions for the study of the experiment.
All test groups were co-cultured for 8 days, and were manually shaken twice daily in the morning and evening, and biogas slurry physicochemical indexes were measured every two days.
Test results
The change of ammonia nitrogen in the culture biogas slurry is shown in figure 4. 3 strains have different degrees of removal capacities for ammonia nitrogen in biogas slurry within 8 days of inoculation. By the 8 th day, the ammonia nitrogen removal rate of 3 microalgae in biogas slurry reaches more than 80%, wherein the effect of chlorella DTG5 strain is best 98.14%, and the algae strains DTG3 and DTY2 are respectively 80.95% and 85.14%. The effect of chlorella on day 21 reported in the patent of CN 109234167a was close to that of the present application. The change of total nitrogen in the culture biogas slurry is shown in figure 5.
The change of total phosphorus in the culture biogas slurry is shown in figure 6. 3 plants have different degrees of uptake and absorption capacity for total phosphorus in biogas slurry within 8 days of inoculation. Wherein, the removal rate of total phosphorus in biogas slurry by chlorella DTG5 strain is 91.93%, which is obviously higher than 55% of the chlorella DTG3 strain and 57.07% of the DTY2 strain. And only 21 days can achieve the similar effect to the treatment of 8 days in the patent. FIG. 7 is a diagram showing comparison of DTG5 sequences with other species. Therefore, the chlorella DTG5 strain obtained through separation and purification has tolerance in biogas slurry with a certain concentration, and has good purification effect and remarkable denitrification and dephosphorization effects. Compared with other two microalgae, the microalgae provided by the invention has stronger purifying capacity and better biogas slurry tolerance, and has more practical application value.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (6)

1. The strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: m2021297, with a preservation address of university of Wuhan, china, a preservation date of 2021, 3 and 29 days, classified and namedChlorella vulgaris（Chlorophyta）DTG5。
2. Use of the pollution-resistant chlorella strain of claim 1 in biogas slurry purification.
3. The use according to claim 2, wherein the culture broth of chlorella DTG5 grown to log phase is grown in a ratio of 1:1 to 1: inoculating the culture medium into diluted biogas slurry in a volume ratio of 32, and culturing.
4. The use according to claim 3, wherein the diluted biogas slurry is a raw biogas slurry which is autoclaved and purified water in a ratio of 1:1-1: 32.
5. The use according to claim 3, wherein the incubation temperature is 25 ℃ ± 1 ℃.
6. The use according to claim 3, wherein the light intensity during cultivation is 40% and the light-to-dark ratio is 12h:12h; shaking twice a day in the morning and evening.
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