Method for treating culture tail water by using phycomycete co-culture system

Abstract

The invention provides a method for treating culture tail water by a phycomycete co-culture system. The method comprises the following steps: step 1, culturing chlorella in a BG11 culture medium; and 2, separating symbiotic bacteria in the chlorella phycosphere environment, and carrying out activation culture. Step 3, carrying out sterile treatment on the chlorella to obtain sterile algae; respectively inoculating sterile algae and symbiotic bacteria into the culture tail water to establish an algae-bacteria co-culture system; the algae and bacteria co-culture system can enhance the activity of metabolic enzymes related to nitrogen and phosphorus in chlorella, so that efficient utilization of nitrogen and phosphorus in tail water is realized.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture tail water treatment, and in particular to a method for treating aquaculture tail water with an algae-bacteria co-culture system. Background Art

[0002] With the rapid development of aquaculture, the issue of aquaculture tailwater discharge has become an important issue in environmental protection. Due to excessive feeding and accumulation of excrement, a large amount of nitrogen and phosphorus remain in aquaculture tailwater. The deterioration of water quality not only causes various diseases to break out in aquaculture objects, but also their metabolites will remain in the environment, causing long-term adverse effects.

[0003] Common methods for aquaculture effluent treatment include physical methods, such as sedimentation, filtration and adsorption, chemical methods, such as the use of chemical agents such as chlorine, ozone and copper sulfate, and biological treatment methods, such as the use of microbial metabolic activity, wetlands and biofilms.

[0004] However, the above methods all have different defects. Physical methods are more effective in removing larger particulate pollutants such as suspended solids and silt, but the removal effect on organic pollutants is limited, and secondary pollution may occur during the treatment process. If the chemical treatment of aquaculture tail water is improper or not completely removed, some chemicals may remain in the water, causing secondary pollution to the environment. The purchase, storage, transportation and use of chemicals all require certain costs, especially for some highly effective but expensive chemicals, the cost of use may be high. Biological treatment of aquaculture tail water takes a certain amount of time to complete the treatment, and the treatment cycle is relatively long. Since the growth and metabolism of microorganisms are greatly affected by temperature, the treatment effect will be affected when the environment fluctuates greatly, and the treatment cycle will be longer.

[0005] Therefore, it is an urgent problem to provide a method for treating aquaculture tail water that is quick in effect, has no secondary pollution and is low in cost. Summary of the invention

[0006] In order to solve the above problems, the present invention provides 1. a method for treating aquaculture tail water by using an algae-bacteria co-culture system, characterized in that it comprises the following steps:

[0007] Step 1: Cultivate Chlorella in BG11 medium at a density of 5×10 6 cells / mL, temperature 24-26°C, light intensity 90-110 μmol / m 2 / s, light-dark ratio 12h:12h, culture 5-7d;

[0008] Step 2, separating the symbiotic bacteria from the Chlorella algae liquid by a plate spreading method, and inoculating the symbiotic bacteria into LB liquid culture medium for activation culture;

[0009] Step 3: sterilize the chlorella in step 1 to obtain sterile algae; inoculate the sterile algae and symbiotic bacteria into the aquaculture tail water respectively, and the density of the sterile algae is (5-15)×10 6 cells / ml, and the density of symbiotic bacteria is (5-25)×10 6 cfu / ml.

[0010] Preferably, the symbiotic bacteria are one or more of Shewanella spp., Bacillus velezensis and Rhodococcus coryneformis from fermented seafood.

[0011] Preferably, the method for separating the symbiotic bacteria by plate coating in step 2 is to dilute the Chlorella solution with sterile water in different gradients, and the density of Chlorella in the different gradient dilutions is 1.0×10 1 -1.0×10 6 cells / mL, take 100 μL of each gradient dilution and spread it evenly on LB solid culture medium, invert and culture at 25-30℃ for 24h-30h, pick out single colonies with different colors and morphologies with an inoculation loop, separate and purify them respectively until a single strain is formed.

[0012] Preferably, the activation culture in step 2 is to place the LB liquid culture medium with the symbiotic bacteria in a constant temperature shaker at a temperature of 25° C.-30° C. and a rotation speed of 120-160 rpm for 2 days.

[0013] Preferably, the aseptic treatment in step three is to add ampicillin, kanamycin sulfate and gentamicin stock solutions to the Chlorella algae liquid, with concentrations of 300 μg / ml, 300 μg / ml and 100 μg / ml respectively.

[0014] The present invention has the following advantages:

[0015] Both Chlorella and microorganisms have strong adaptability and survival ability and grow fast. The two can establish a co-culture system with good stability, quickly adapt to various environments and operate stably for a long time, and synergistically absorb nitrogen, phosphorus and other nutrients in the tail water to achieve the purpose of rapid purification.

[0016] The establishment of an algae-bacteria co-culture system can quickly convert nutrients and organic matter into recyclable resources, produce less waste and pollutants, significantly reduce the pollution pressure on surrounding water bodies, and is highly environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 The figure shows the colony morphology and growth curve.

[0019] Figure 2 This is a diagram of DNA identification and phylogenetic analysis.

[0020] Figure 3 Gel electrophoresis diagram.

[0021] Figure 4 This is a graph showing the growth indicators of Chlorella in the co-culture system of Chlorella and different strains.

[0022] Figure 5 This is the bacterial growth in the co-culture system of Chlorella and different strains.

[0023] Figure 6 This is a graph showing the growth index of Chlorella in the co-culture system with different algae-bacteria ratios.

[0024] Figure 7 This is a graph of the growth indicators of Chlorella in different algae-bacteria co-culture systems.

[0025] Figure 8 This is a diagram showing the purification effect of different algae-bacteria co-culture systems on aquaculture tail water.

[0026] Fig. 9 This is a diagram showing the purification effect of the Chlorella-Bacillus Velez co-culture system on artificial aquaculture tail water.

[0027] Fig.10 This is a graph showing the changes in enzyme activities related to nitrogen absorption by Chlorella in the Chlorella-Bacillus Velez co-culture system. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] The chlorella used in the following test examples is Chlorella vulgaris, hereinafter referred to as Cv, purchased from Shanghai Guangyu Biotechnology Co., Ltd.

[0030] Test Example 1

[0031] Step 1: Place the Chlorella in BG11 culture medium and culture it in a light incubator. The density of the Chlorella is 5×10 6 cells / mL, the culture temperature was (25±1)℃, the light intensity was (100±10)μmol / m / s, the light-dark ratio was 12h:12h, and the culture was carried out for 7 days. At this time, the density of Chlorella was 1.0×10 7 cells / mL.

[0032] Step 2: Use sterile water to dilute the Chlorella solution in different gradients. The density of Chlorella in different gradient dilutions is 1.0×10 1 -1.0×10 6 cells / mL, 100 μL of each gradient dilution was evenly spread on LB solid medium, inverted and cultured at 30°C for 24 h, and three bacterial strains were separated based on color and morphological differences, such as Figure 1 As shown, they are named S1-S3. The following identifications were performed on the S1-S3 strains:

[0033] 1) After plate streaking purification, DNA of three bacterial strains was extracted and 16SrDNA sequence was amplified with a fragment size of 1500 bp for DNA identification and phylogenetic analysis. Figure 2 shown.

[0034] 2) Spot samples on agarose gel for electrophoresis verification. From left to right, they are S1, S2, and S3. Figure 3 shown.

[0035] 3) Physiological and biochemical characteristics are shown in Table 1.

[0036] Table 1 Physiological and biochemical characteristics

[0037]

[0038]

[0039] Combination Figure 1-3 As shown in Table 1, strains S1, S2 and S3 were identified as Alishewanella jeotgali, Bacillus velezensis and Rhodococcus corynebacterioides, respectively. They are referred to as Aj, Bv and Rc, respectively.

[0040] Step three, add ampicillin, kanamycin sulfate and gentamicin stock solutions to the Chlorella liquid at concentrations of 300 μg / ml, 300 μg / ml and 100 μg / ml, respectively, and treat for 3 times, with an interval of 2 days between each treatment, for 6 days, and culture for 1 generation to eliminate the adverse effects of antibiotics on Chlorella.

[0041] The strains S1, S2 and S3 were mixed with Chlorella and inoculated into the aquaculture tail water at an inoculation density of 5×10 6 cells / mL,5×10 6 cfu / mL, with the blank group as the control. The cell density, chlorophyll content, maximum light energy conversion efficiency, maximum photosynthetic rate, half-saturation light intensity, initial slope of the fast light curve and bacterial growth of Chlorella were measured every 2 days. The results are as follows Figure 4-5 shown.

[0042] according to Figure 4-5 It can be seen that the three strains can promote the growth of Chlorella. On the 8th day, the Fv / Fm of the algae-bacteria system was significantly higher than that of the pure culture system of Chlorella (P<0.05). In the algae-bacteria co-culture system constructed by Bacillus Velezii and Chlorella, the algae cell density was 29.65×10 6 cells / mL, and the Chla content was 5.48 mg / L, which was significantly higher than that of other algae-bacteria systems (P<0.05). The ETRmax of Chlorella in the C.v+Bv system rose to 389 at the end of culture, which was significantly higher than that of the pure culture system of Chlorella (P<0.01). On the 8th day of culture, the IK and α of Chlorella cells in the C.v+Bv system were 1738 and 0.18, respectively, which were significantly better than those of other systems (P<0.05). The concentration of Bacillus Velezii after 8 days was 1.65×10 7 cfu / mL, which is 3.3 times higher than the initial concentration, indicating that the strain of bacteria has a strong adaptability to Chlorella. Comparing the growth of Chlorella and the three bacterial strains, Bacillus Velezii is more suitable for constructing an algae-bacteria co-culture system with Chlorella.

[0043] The sterile algae and Bacillus Velezii were inoculated into the aquaculture tail water according to the inoculation ratio listed in Table 2 and cultured for 8 days. Samples were taken every 2 days to measure the cell density, Chla content and Fv / Fm of Chlorella. The results are shown in Table 2. Figure 6 shown.

[0044] Table 2 Initial inoculation density of Chlorella and Bv strains

[0045] Algae to fungus ratio <![CDATA[Chlorella × 10 6 cells / mL]]> <![CDATA[Strain × 10 6 cfu / mL]]> 1:0 5 0 1:1 5 5 1:3 5 15 1:5 5 25 3:1 15 5

[0046] According to Table 2 and Figure 6It can be seen that under the same culture conditions, when the algae-bacteria inoculation ratio was 1:3, the Chla content of Chlorella reached 5.68 mg / L on the 8th day, and the algae cell density was 33.05×10 6 cells / mL, which was significantly higher than that of other ratio combinations (P<0.05).

[0047] Chlorella and the isolated symbiotic bacteria were used to construct a single algae-single bacteria and a single algae-composite bacteria co-culture system and inoculated into the aquaculture tail water, as shown in Table 3. Samples were taken every 2 days to measure the growth index of Chlorella and the tail water nitrogen, phosphorus and other indicators, and the effects of different algae-bacteria systems on the growth of Chlorella and the treatment effect on aquaculture tail water were analyzed. The results are shown in Table 3. Figure 7-8 .

[0048] Table 3 Initial inoculation density of algae-bacteria co-culture system

[0049]

[0050] Combining Table 3 and Figure 7-8 It can be seen that after 8 days of cultivation, the cell density of Chlorella vulgaris in the C.v+Bv co-culture system was 15.87×10 6 cells / mL, the Chla content of Chlorella was 3.95 mg / L, and its effect on aquaculture effluent treatment was significantly higher than that of other algae-bacteria systems (P<0.01).

[0051] Using the pure culture of sterile Chlorella as a control, Chlorella and Bacillus Velez were inoculated at a density of 1:3 (5×10 6 cells / mL,15×10 6 cfu / mL) to construct an algae-bacteria co-culture system, and culture for 10 days. The growth index of Chlorella, the concentration of nitrogen and phosphorus in the water, and the activity of nitrogen and phosphorus metabolic enzymes of Chlorella were measured every other day. The results are shown in Figure 9-10 .

[0052] Depend on Figure 9-10 It can be seen that when cultured for 6-8 days, the AKP and ACP activities of the two systems were strong, and the highest enzyme activities of Chlorella in C.v+Bv were 7.6 King's units / 100mL and 6.53 King's units / 100mL, respectively, which were significantly better than the pure culture system of Chlorella (P<0.05).

[0053] Conclusion: Three symbiotic bacteria were isolated and purified from the algal environment of stably cultured Chlorella vulgaris, and were identified as Heteroshewanella spp., Bacillus velez and Rhodococcus spp. The results showed that Bacillus velez was more suitable for constructing an algae-bacteria co-culture system with Chlorella vulgaris.

[0054] The optimal inoculation ratio of Chlorella to symbiotic bacteria was 1:3. The growth index of Chlorella and the purification effect of aquaculture tail water in the algae-bacteria co-culture system constructed by Chlorella vulgaris and Bacillus Velez were significantly better than those of other algae-bacteria and pure culture systems (P<0.01).

[0055] The algae-bacteria co-culture system constructed by Chlorella vulgaris-Velez spores has a significantly better treatment effect on artificial aquaculture tail water and nitrogen and phosphorus metabolic enzyme activities than the pure culture system of Chlorella (P<0.05). The algae-bacteria co-culture system can enhance the activity of Chlorella's nitrogen and phosphorus metabolic enzymes, thereby achieving efficient utilization of nitrogen and phosphorus in tail water.

[0056] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for treating aquaculture tail water using an algae-bacteria co-culture system, characterized in that: The following steps are involved: Step 1: Cultivate Chlorella in BG11 medium at a density of 5×10 6 cells / mL, temperature 24-26°C, light intensity 90-110 μmol / m 2 / s, light-dark ratio 12h:12h, culture 5-7d; Step 2, separating the symbiotic bacteria from the Chlorella algae liquid by a plate spreading method, and inoculating the symbiotic bacteria into LB liquid culture medium for activation culture; Step 3: sterilize the chlorella in step 1 to obtain sterile algae; inoculate the sterile algae and symbiotic bacteria into the aquaculture tail water respectively, and the density of the sterile algae is (5-15)×10 6 cells / ml, and the density of symbiotic bacteria is (5-25)×10 6 cfu / ml.

2. The method for treating aquaculture tail water by using an algae-bacteria co-cultivation system according to claim 1, characterized in that: The symbiotic bacteria are one or more of the fermented seafood Heswanella, Bacillus Velezii and Rhodococcus coryneformis.

3. The method for treating aquaculture tail water by using an algae-bacteria co-cultivation system according to claim 1, characterized in that: The method for separating the symbiotic bacteria by plate coating in step 2 is to dilute the Chlorella solution with sterile water in different gradients, and the density of Chlorella in the different gradient dilutions is 1.0×10 1 -1.0×10 6 cells / mL, take 100 μL of each gradient dilution and spread it evenly on LB solid culture medium, invert and culture at 25-30℃ for 24h-30h, pick out single colonies with different colors and morphologies with an inoculation loop, separate and purify them respectively until a single strain is formed.

4. The method for treating aquaculture tail water by using an algae-bacteria co-cultivation system according to claim 1, characterized in that: The activation culture in step 2 is to place the LB liquid culture medium with the symbiotic bacteria in a constant temperature shaker at a temperature of 25°C-30°C and a rotation speed of 120-160 rpm for 2 days.

5. The method for treating aquaculture tail water by using an algae-bacteria co-cultivation system according to claim 1, characterized in that: The aseptic treatment in step 3 is to add ampicillin, kanamycin sulfate and gentamicin stock solutions to the Chlorella algae liquid, with concentrations of 300 μg / ml, 300 μg / ml and 100 μg / ml respectively.

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