Bacteria-algae co-immobilized material and application thereof in red tide treatment of prorocentrum donghaiense

Abstract

The invention discloses a bacterium-alga co-immobilized material and application thereof in red tide treatment of prorocentrum donghaiense, and belongs to the field of microorganisms for harmful red tide treatment. According to the invention, alteromonas algicidal bacteria and autotrophic non-red-tide microalgae Tetraselmis are co-immobilized on the same carrier in an adsorption manner to form a bacteria-algae co-immobilized material. The bacterium-algae co-immobilization material has high algae-lysing performance on prorocentrum donghaiense red tide, the algae-lysing rate is 100% within 36 hours, and the algae-lysing rate is 84.07% after the bacterium-algae co-immobilization material is repeatedly used for four times. The bacteria-algae co-immobilized material has the characteristics of no toxicity, strong water absorption, high stability, reusability and the like, and can be effectively used for treating harmful algal blooms of red tide algae such as prorocentrum donghaiense.

Description

Technical Field

[0001] The present invention relates to the field of environmental microbiology, and particularly to a material for co-immobilization of bacteria and algae and its application in the treatment of red tides caused by Technical Background

[0002] Prorocentrum donghaiense belongs to a type of single-celled phytoplankton in the class Dinophyceae. Its massive reproduction can cause the seawater to change color and form red tides, which have a serious impact on the marine ecosystem and fisheries. For the prevention and control of red tides, in addition to traditional physical and chemical methods, bioremediation technologies, especially immobilized microorganism technology, have become a research hotspot due to their high efficiency, stability, and relatively high biosafety. Immobilized microorganism technology can immobilize microorganisms on specific carriers through adsorption or encapsulation, etc. It not only provides an attachment and protection space for microorganisms but also avoids their direct contact with pollutants, reducing the risk of introducing new ecological problems.

[0003] The immobilized bacteria technology immobilizes bacteria that can inhibit or kill red tide algae, i.e., algicidal bacteria, on specific carriers through physical or chemical methods, enabling them to continuously exert their algicidal function within a certain period of time, thereby achieving effective prevention and control of red tides. However, as the usage time extends, the algicidal bacteria on the carrier may experience a decline in their biological activity due to the depletion of nutrients, the accumulation of their own metabolites, and changes in the surrounding environmental conditions, thereby affecting the persistence and high efficiency of their algicidal effect.

[0004] The co-immobilization system of bacteria and algae has higher algicidal efficiency compared to single immobilized bacteria. This advantage stems from the synergistic effect between autotrophic algae and algicidal bacteria within the system, which can jointly construct a stable microecosystem. In this system, algicidal bacteria can inhibit the growth of red tide algae, while autotrophic algae increase the dissolved oxygen content in the water through photosynthesis, thereby improving water quality and promoting the restoration of the ecosystem. In addition, there is a positive interaction between bacteria and algae: the metabolic activities of algicidal bacteria can provide carbon dioxide and other nutrients for autotrophic algae, and the oxygen released by algae photosynthesis is also convenient for bacteria to absorb and support their metabolic processes, thus realizing mutualism between bacteria and algae, which provides the possibility for the long-term and highly efficient algicidal effect of the co-immobilization system of bacteria and algae. Although the immobilized bacteria-algae system demonstrates significant advantages, it still faces challenges in practical applications. The development of the co-immobilization technology of bacteria and algae provides a new perspective for red tide prevention and control. Summary of the Invention

[0005] The present invention provides a bacteria-algae co-immobilized material and its application in the treatment of red tides caused by Prorocentrum donghaiense. In the present invention, an algicidal bacterium and autotrophic bait microalgae are co-immobilized on a sponge carrier by an adsorption method to form a bacteria-algae co-immobilized material, and the bacteria-algae co-immobilized material is used to treat the red tide caused by Prorocentrum donghaiense, and the material can be reused. Among them, the autotrophic bait microalgae provide oxygen and organic nutrients for the algicidal bacteria in the co-immobilized material through photosynthesis to support the growth and reproduction of the algicidal bacteria, so as to achieve a continuous algicidal effect.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a bacteria-algae co-immobilized material, which is obtained by co-immobilizing an algicidal bacterium and autotrophic non-red tide microalgae on the same carrier by an adsorption method; Further, the algicidal bacterium is an algicidal bacterium of the genus Alteromonas; Further, the autotrophic non-red tide microalgae are Tetraselmis; Further, the carrier is a polyurethane sponge.

[0007] The second aspect of the present invention provides a preparation method of the above-mentioned bacteria-algae co-immobilized material, including the following steps: preparing a mixed suspension of an algicidal bacterium and autotrophic non-red tide microalgae, and then adding the pretreated carrier to the mixed suspension for adsorption and immobilization to obtain a bacteria-algae co-immobilized material; Further, the cell density ratio of the algicidal bacterium to the autotrophic non-red tide microalgae in the mixed suspension is 20:1; Further, the conditions for the adsorption and immobilization are: adsorption time 24 h, adsorption temperature 25 °C, adsorption rotation speed 120 rpm, and the volume ratio of the pretreated carrier to the mixed suspension is 1:7.

[0008] The third aspect of the present invention provides the application of the above-mentioned bacteria-algae co-immobilized material in the treatment of red tides; Further, the red tide is a red tide caused by Prorocentrum donghaiense; Further, the method of the application is: taking 3 pieces of bacteria-algae co-immobilized materials with a volume of 1 cm 3 and adding them to 100 mL of the exponential-phase culture of Prorocentrum donghaiense for algicidal treatment.

[0009] The main effects and advantages of the present invention are as follows: 1. A bacteria-algae co-immobilized material is prepared by co-immobilizing the algicidal bacterium FDHY-03 and autotrophic non-red tide microalgae Tetraselmis on the same carrier by an adsorption method. The algicidal rate is 100% after 36 h. Then, the fresh Prorocentrum donghaiense algal solution is replaced every 48 h, and the algicidal rate is 84.07% after repeating the algicidal treatment 4 times.

[0010] 2. Both the algicidal bacterium FDHY-03 and the autotrophic non-red tide microalga Tetraselmis are derived from the marine ecological environment. Selecting polyurethane sponge as the carrier has the characteristics of low density and non-toxic environmental protection, which is convenient for deployment and recovery in the pilot waters and supports repeated use. It has good application prospects in controlling the red tide of Prorocentrum donghaiense.

[0011] 3. By co-immobilizing the algicidal bacterium FDHY-03 and the autotrophic non-red tide microalga Tetraselmis on the same carrier and utilizing the synergistic effect between the two, a symbiotic system of bacteria and algae with long-term and stable algicidal efficacy is constructed, providing a new idea and new approach for the red tide disaster prevention and mitigation system. Description of the Drawings

[0012] Figure 1 It is a microstructural observation diagram of the polyurethane sponge in Example 1.

[0013] Figure 2 It is a microstructural observation diagram of the bacteria-algae co-immobilized material in Example 1.

[0014] Figure 3 It is the algicidal effect diagram of different carrier materials on Prorocentrum donghaiense in Example 2.

[0015] Figure 4 It is the algicidal effect diagram of the bacteria-algae co-immobilized material obtained under different immobilization conditions on Prorocentrum donghaiense in Example 3.

[0016] Figure 5 It is the algicidal effect diagram of the bacteria-algae co-immobilized material under 4 repeated uses in Example 4. Detailed Embodiments

[0017] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. Unless otherwise specified, the methods involved in the embodiments are common methods, and the reagents involved are commercially available conventional reagents.

[0018] The algicidal bacterium involved in the present invention is Alteromonas macleodii FDHY-03, whose taxonomic name is Alteromonas macleodii. The accession number of its 16S rRNA gene in GenBank is MH919332. This strain has been disclosed in the literature

SHI X G, LIU L M, LI Y, et al. Isolation of an algicidal bacterium and its effects against the harmful-algal-bloom dinoflagellate Prorocentrum donghaiense (Dinophyceae) [J]. Harmful Algae, 2018, 80: 72-9.

[0019] The formula of the 2216E solid medium involved in the present invention is: containing 5 g of peptone, 1 g of yeast extract powder, 0.01 g of ferric phosphate, 30 g of sea salt, 15 g of agar powder in every 1 L volume, and the rest is pure water; pH = 7.2; sterilized at 121 °C for 20-25 min.

[0020] The formula of the optimized medium involved in the present invention is: containing 42.5 g of peptone, 4.6 g of sucrose, 0.01 g of ferric phosphate, 30 g of sea salt in every 1 L volume, and the rest is pure water; pH = 7.5; sterilized at 121 °C for 20-25 min.

[0021] The preparation method of the FDHY-03 exponential-phase culture involved in the present invention is: taking out the Alteromonas macleodii FDHY-03 strain from the -80 °C refrigerator, inoculating it on the 2216E solid medium, culturing it in a 25 °C biochemical incubator for 24 h to obtain an activated strain; picking a single colony of the activated strain and inoculating it into 30 mL of the optimized medium, culturing it at 25 °C and 150 rpm for 24 h to obtain a primary seed solution; inoculating the primary seed solution into 100 mL of the optimized medium at an inoculation amount of 1 vol%, and culturing it at 25 °C and 150 rpm until the exponential growth phase to obtain the FDHY-03 exponential-phase culture.

[0022] The present invention does not have special limitations on the source of the red tide alga Prorocentrum donghaiense involved, and it can be obtained by using conventional commercially available products in the art or collecting from coastal areas according to conventional methods in the art. Specifically, the Prorocentrum donghaiense in the present invention is provided by the Key Laboratory of Marine Environmental Science, Xiamen University. The Prorocentrum donghaiense is inoculated into the modified f / 2 medium and cultured in a light incubator at a temperature of 20 °C, a light intensity of 80 μmol photons·m -2 ·s -1 and a light-dark cycle of 14 h:10 h until the exponential growth phase, obtaining the exponential-phase culture of Prorocentrum donghaiense.

[0023] The present invention does not have special limitations on the source of the alga Tetraselmis subcordiformis involved, and it can be obtained by using conventional commercially available products in the art or collecting from coastal areas according to conventional methods in the art. Specifically, the Tetraselmis subcordiformis in the present invention is provided by the Key Laboratory of Marine Biotechnology, Jiangsu Ocean University. The Tetraselmis subcordiformis is inoculated into the modified f / 2 medium and cultured in a light incubator at a temperature of 20 °C, a light intensity of 80 μmol photons·m -2 ·s -1 and a light-dark cycle of 14 h:10 h until the exponential growth phase, obtaining the exponential-phase culture of Tetraselmis subcordiformis.

[0024] The formula of the modified f / 2 medium involved in the present invention is as follows: in each 1 L volume, it contains 1 mL of vitamin solution, 1 mL of metal salt solution, 1 mL of NaNO 3 solution, 1 mL of NaH 2 PO 4 ·H 2 O solution, and the rest is natural seawater with a salinity of 30‰; sterilized at 121 °C for 30 min. Among them, the formula of the vitamin solution is: in each 100 mL volume, it contains 10 mg of vitamin B 1 , 0.05 mg of vitamin B 12 , 0.05 mg of vitamin B 7 , and the rest is natural seawater with a salinity of 30‰; the formula of the metal salt solution is: in each 1 L volume, it contains 9.8 mg of CuSO 4 ·5H 2 O, 22 mg of ZnSO 4 ·7H 2 O, 10 mg of CoCl·6H 2 O, 180 mg of MnCl 2 ·4H 2 O, 6.3 mg of Na 2 MoO 4 ·2H 2O, 4.36 g of EDTA-2Na, 3.15 g of FeCl 3 ·6H 2 O, and the rest is natural seawater with a salinity of 30‰; NaNO 3 The solution formula is: containing 7.5 g of NaNO in every 100 mL volume 3 , and the rest is natural seawater with a salinity of 30‰; NaH 2 PO 4 ·H 2 O solution formula is: containing 0.5 g of NaH 2 PO 4 ·H 2 O, and the rest is natural seawater with a salinity of 30‰; The natural seawater needs to be filtered with a 0.22 μm filter membrane before use.

[0025] The carrier material involved in the present invention selects polyurethane sponge with a density of 0.13 g / cm 3 , purchased from Quanzhou Shengrong Shoe Material Co., Ltd.; its pretreatment steps are: cutting the polyurethane sponge into small cubes with a specification of 1 cm × 1 cm × 1 cm, washing it 3 times with deionized water, then placing it in an oven at 65°C and drying it for 24 h, then autoclaving it at 121°C for 20 min, and finally placing it in the oven at 65°C again and drying it for 24 h to obtain the pretreated polyurethane sponge.

[0026] The calculation method of the algicidal rate involved in the present invention: Algicidal rate (%) = (1 - algal cell concentration in the experimental group / algal cell concentration in the control group) × 100%.

[0027] Example 1: Preparation of the immobilized carrier Preparation of the algicidal bacteria - Tetraselmis suecica co-immobilized carrier (also known as "bacteria-algae co-immobilized material"): Centrifuge the exponential-phase culture of FDHY-03 at 6000 rpm for 6 min to obtain the algicidal bacteria cell precipitate; centrifuge the exponential-phase culture of Tetraselmis suecica at 6000 rpm for 6 min to obtain the Tetraselmis suecica cell precipitate; resuspend the algicidal bacteria cell precipitate and the Tetraselmis suecica cell precipitate in the modified f / 2 medium to obtain the algicidal bacteria - Tetraselmis suecica mixed suspension; add the pretreated polyurethane sponge to the algicidal bacteria - Tetraselmis suecica mixed suspension, take it out after adsorbing at 25°C and 120 rpm for 12 h to obtain the algicidal bacteria - Tetraselmis suecica co-immobilized carrier. Among them, the final cell densities of the algicidal bacteria cell precipitate and the Tetraselmis suecica cell precipitate in the algicidal bacteria - Tetraselmis suecica mixed suspension are 3.5×10 7 cell / ml and 1.75×10 6 cell / ml respectively; the volume ratio of the pretreated polyurethane sponge to the algicidal bacteria - Tetraselmis suecica mixed suspension is 1:10.

[0028] Preparation of immobilized carrier of algicidal bacteria: The exponential-phase culture of FDHY-03 was centrifuged at 6000 rpm for 6 min to obtain the precipitate of algicidal bacteria cells; the precipitate of algicidal bacteria cells was resuspended in the modified f / 2 medium to obtain the suspension of algicidal bacteria; the pretreated polyurethane sponge was added to the suspension of algicidal bacteria, and after adsorption at 25 °C and 120 rpm for 12 h, it was taken out to obtain the immobilized carrier of algicidal bacteria. Among them, the final cell density of the precipitate of algicidal bacteria cells in the suspension of algicidal bacteria was 3.5×10 7 cell / ml; the volume ratio of the pretreated polyurethane sponge to the suspension of algicidal bacteria was 1:10.

[0029] Preparation of immobilized carrier of Tetraselmis suecica: The exponential-phase culture of Tetraselmis suecica was centrifuged at 6000 rpm for 6 min to obtain the precipitate of Tetraselmis suecica cells; the precipitate of Tetraselmis suecica cells was resuspended in the modified f / 2 medium to obtain the suspension of Tetraselmis suecica; the pretreated polyurethane sponge was added to the suspension of Tetraselmis suecica, and after adsorption at 25 °C and 120 rpm for 12 h, it was taken out to obtain the immobilized carrier of Tetraselmis suecica. Among them, the final cell density of the precipitate of Tetraselmis suecica cells in the suspension of Tetraselmis suecica was 1.75×10 6 cell / ml; the volume ratio of the pretreated polyurethane sponge to the suspension of Tetraselmis suecica was 1:10.

[0030] Figure 1 Figure is the microstructural observation diagram of the polyurethane sponge. It can be seen from the figure that the polyurethane sponge contains a large number of dense and regular pore structures, which is beneficial to the attachment of algicidal bacteria and Tetraselmis suecica, and can provide sufficient space for their growth and reproduction.

[0031] Figure 2 Figure is the microstructural observation diagram of the co-immobilized carrier of algicidal bacteria - Tetraselmis suecica. It can be seen from the figure that a large number of algicidal bacteria and Tetraselmis suecica are contained in the pore structure of the pretreated polyurethane sponge, indicating that the algicidal bacteria and Tetraselmis suecica have been successfully adsorbed on the carrier material.

[0032] Example 2: Algicidal effect of the co-immobilized carrier of algicidal bacteria - Tetraselmis suecica on Prorocentrum donghaiense In the experimental group, the pretreated polyurethane sponge (blank carrier), the co-immobilized carrier of algicidal bacteria - Tetraselmis suecica, the immobilized carrier of algicidal bacteria, and the immobilized carrier of Tetraselmis suecica in Example 1 were respectively put into the exponential-phase culture of Prorocentrum donghaiense, and 3 carriers were added to every 100 mL of the exponential-phase culture of Prorocentrum donghaiense; another group of exponential-phase culture of Prorocentrum donghaiense without adding any carrier was prepared as the control group. The whole algicidal experiment was carried out in a light incubator at a temperature of 20 °C, a light intensity of 80 μmol photons·m -2 ·s -1 and a photoperiod of 14:10 h. The first algicidal cycle was set to 48 hours, and then the fresh exponential-phase culture of Prorocentrum donghaiense was replaced every 48 hours, and the algal cell density and algicidal rate were calculated. The results are shown in Figure 3 .

[0033] From Figure 3 It can be seen that the blank carrier has little effect on the growth of *Prorocentrum donghaiense*, while the co-immobilized carrier of algicidal bacteria and *Tetraselmis suecica*, the immobilized carrier of algicidal bacteria, and the immobilized carrier of *Tetraselmis suecica* all show a certain inhibitory effect on the growth of *Prorocentrum donghaiense*; among them, the algicidal effect of the co-immobilized carrier of algicidal bacteria and *Tetraselmis suecica* on *Prorocentrum donghaiense* is significantly higher than that of the immobilized carrier of algicidal bacteria and the immobilized carrier of *Tetraselmis suecica*. The algicidal rate reaches 100% at 48 h in the first cycle and 97.87% at 48 h in the second cycle.

[0034] Example 3: Optimization of the immobilization process of the co-immobilized carrier of algicidal bacteria and *Tetraselmis suecica* by the single-factor method To obtain the co-immobilized carrier of algicidal bacteria and *Tetraselmis suecica* with the best performance, the immobilization process was optimized. Single-factor experiments were carried out on different adsorption times (6 h, 12 h, 24 h, 36 h, 48 h), adsorption temperatures (15 °C, 20 °C, 25 °C, 30 °C), adsorption rotation speeds (90 rpm, 120 rpm, 150 rpm, 180 rpm), and adsorption solid-liquid ratios (1:2, 1:5, 1:7, 1:10, 1:13). The prepared co-immobilized carrier of algicidal bacteria and *Tetraselmis suecica* was put into the exponential-phase culture of *Prorocentrum donghaiense*, and 3 carriers were added to every 100 mL of the exponential-phase culture of *Prorocentrum donghaiense*. The optimal conditions were determined through two algicidal evaluations. The whole algicidal experiment was carried out in a light incubator at a temperature of 20 °C, a light intensity of 80 μmol photons·m -2 ·s -1 and a light cycle of 14:10 h: The first evaluation was to sample and count after 12 h, and the second was to sample and count again 36 h after replacing the fresh *Prorocentrum donghaiense* culture.

[0035] The results are as Figure 4 shown. At an adsorption time of 24 h ( Figure 4 A), an adsorption temperature of 25 °C ( Figure 4 B), and an adsorption rotation speed of 120 rpm ( Figure 4 C), the algicidal effects of the two times are better than those of other treatment groups; it can be seen from the change in the algicidal rate of *Prorocentrum donghaiense* with the adsorption solid-liquid ratio ( Figure 4 D) that with the increase in the volume of the adsorption liquid in the adsorption solid-liquid ratio, the algicidal rate does not change significantly during the first algicidal process, while the algicidal rate reaches the maximum value at a solid-liquid ratio of 1:7 during the second algicidal process and then tends to be stable.

[0036] Finally, the optimal immobilization conditions obtained through the single-factor experiment are: adsorption time 24 h, adsorption temperature 25 °C, adsorption rotation speed 120 rpm, and solid-liquid ratio 1:7.

[0037] Example 4: Evaluation of the Reusability of the Optimized Immobilized Carrier of Algae-lysing Bacteria and Tetraselmis suecica Centrifuge the exponential-phase culture of FDHY-03 at 6000 rpm for 6 min to obtain the precipitate of algae-lysing bacteria cells; centrifuge the exponential-phase culture of Tetraselmis suecica at 6000 rpm for 6 min to obtain the precipitate of Tetraselmis suecica cells; resuspend the precipitate of algae-lysing bacteria cells and the precipitate of Tetraselmis suecica cells in the modified f / 2 medium to obtain the mixed suspension of algae-lysing bacteria and Tetraselmis suecica; add the pretreated polyurethane sponge into the mixed suspension of algae-lysing bacteria and Tetraselmis suecica, take it out after adsorbing at 25 °C and 120 rpm for 24 h to obtain the immobilized carrier of algae-lysing bacteria and Tetraselmis suecica. Among them, the final cell densities of the precipitate of algae-lysing bacteria cells and the precipitate of Tetraselmis suecica cells in the mixed suspension of algae-lysing bacteria and Tetraselmis suecica are 3.5×10 7 cell / ml and 1.75×10 6 cell / ml respectively; the volume ratio of the pretreated polyurethane sponge to the mixed suspension of algae-lysing bacteria and Tetraselmis suecica is 1:7.

[0038] Put the prepared immobilized carrier of bacteria and algae into the exponential-phase culture of 100 mL of Prorocentrum donghaiense, and add 3 carriers to every 100 mL of the exponential-phase culture of Prorocentrum donghaiense; prepare another group of exponential-phase culture of Prorocentrum donghaiense without adding any carrier as the control group. The whole algae-lysing experiment is carried out in a light incubator at a temperature of 20 °C, a light intensity of 80 μmol photons·m -2 ·s -1 and a photoperiod of 14:10 h. The first algae-lysing cycle is set to 36 hours, and then replace the fresh culture of Prorocentrum donghaiense every 48 hours, and calculate its algae-lysing rate.

[0039] The results are as Figure 5 shown. After 4 repeated algae-lysing experiments, the algae-lysing rate of the immobilized carrier of algae-lysing bacteria and Tetraselmis suecica can still reach 84.07%. Therefore, the immobilized carrier of algae-lysing bacteria and Tetraselmis suecica can be used for the treatment of Prorocentrum donghaiense red tide.

Claims

1. A bacteria-algae co-immobilization material, characterized in that: The bacteria-algae co-immobilized material is obtained by co-immobilizing algae-lysing bacteria and autotrophic non-red tide microalgae on the same carrier by adsorption.

2. The bacteria-algae co-immobilization material according to claim 1, characterized in that: The algae-lytic bacteria is an algae-lytic bacteria of the genus Alteromonas.

3. The bacteria-algae co-immobilization material according to claim 1, characterized in that: The autotrophic non-red tide microalgae is Tetraselmis.

4. The bacteria-algae co-immobilization material according to claim 1, characterized in that: The carrier is a polyurethane sponge.

5. The method for preparing the bacteria-algae co-immobilization material according to claim 1, characterized in that: A mixed suspension of algicidal bacteria and autotrophic non-red tide microalgae is prepared, and then the pretreated carrier is added to the mixed suspension for adsorption and fixation to obtain a bacteria-algae co-immobilized material.

6. The preparation method according to claim 5, characterized in that: The cell density ratio of algicidal bacteria to autotrophic non-red tide microalgae in the mixed suspension is 20:

1.

7. The preparation method according to claim 5, characterized in that: The adsorption and fixation conditions are as follows: adsorption time 24h, adsorption temperature 25°C, adsorption speed 120rpm, and the volume ratio of the pretreated carrier to the mixed suspension is 1:

7.

8. Use of the bacteria-algae co-immobilization material as claimed in claim 1 in red tide control.

9. The use according to claim 8, characterized in that: The red tide described is the Prorocentrum dinoflagellates red tide in the East China Sea.

10. The use according to claim 9, characterized in that: Take 3 pieces of 1cm in size 3 The bacteria-algae co-immobilized material was added to 100 mL of the exponential stage culture of Prorocentrum donghaiense for algae lysis treatment.

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