Method for reducing inhibition of free nitrous acid on toxicity of microalgae

Abstract

The invention discloses a method for reducing inhibition of free nitrous acid on toxicity of microalgae, and belongs to the technical field of biology and new medicine. In order to solve the problem that free nitrous acid (FNA) in a microalgae-nitrifying bacteria co-culture system destroys a microalgae photosynthetic system and inhibits growth, alpha-ketoglutaric acid with the final concentration of 0.5-1.0 mM is exogenously added into the microalgae-nitrifying bacteria co-culture system, so that carbon and nitrogen metabolism of microalgae is regulated and controlled, the tolerance of the microalgae to FNA is enhanced, the photosynthetic efficiency and biomass of the microalgae are remarkably improved, and the microalgae-nitrifying bacteria co-culture system can be used for preparing the microalgae-nitrifying bacteria co-culture system. The ammonia nitrogen removal rate and the system stability are improved; the method is easy and convenient to operate, low in cost, suitable for high-ammonia-nitrogen wastewater treatment and capable of assisting long-term stable operation of a phycomycete symbiotic system.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biology and new medicine technology, and particularly relates to a method for reducing the toxicity inhibition of free nitrous acid on microalgae. BACKGROUND

[0002] Microalgae biotechnology has a wide application prospect in wastewater treatment and resource utilization due to its high nitrogen and phosphorus absorption and conversion capacity and carbon fixation efficiency. In recent years, the co-culture system constructed by coupling microalgae and nitrifying or partial nitrifying bacteria has shown significant advantages in relieving the toxicity of high-concentration ammonia nitrogen on microalgae, promoting the transformation of nitrogen and phosphorus in wastewater into high-value products (such as protein and oil), and reducing the energy consumption of aeration. However, the stable operation of the system is severely restricted by free nitrous acid (FNA) produced in the nitrification process. FNA is easy to accumulate under acidic conditions, can penetrate the microbial cell membrane, increase the proton permeability, reduce the intracellular pH, interfere with the ATPase function and damage the photosynthetic system, thereby strongly inhibiting the growth of microalgae and even leading to the collapse of the entire symbiotic system.

[0003] The main strategies to alleviate the inhibition of FNA at present include improving the activity of nitrite oxidizing bacteria or maintaining the alkaline environment pH to reduce the concentration of FNA. However, FNA as an intermediate product of nitrification is difficult to completely avoid, and even at a low concentration (such as 1.0 mg N / L), it still causes significant inhibition to microalgae. In addition, maintaining alkaline conditions in high-ammonia-nitrogen (more than 45 mg / L) wastewater will promote the accumulation of toxic free ammonia (FA), thereby causing new inhibition problems; therefore, it is urgent to explore a more effective method to avoid the influence of free nitrous acid on microalgae, thereby improving the stability of the symbiotic system of microalgae and nitrifying bacteria. SUMMARY

[0004] In view of some deficiencies in the prior art, the present application provides a method for reducing the toxicity inhibition of FNA on microalgae, specifically, a method for regulating microalgae nitrogen metabolism and enhancing its tolerance to FNA by exogenous addition of alpha-ketoglutarate; alpha-ketoglutarate is a precursor of various amino acid synthesis and a key intermediate metabolite of the tricarboxylic acid cycle, and plays a core regulatory role in carbon and nitrogen metabolism balance. The present application proves by experiments that the addition of trace alpha-ketoglutarate in the microalgae-nitrifying bacteria co-culture system can significantly reduce the inhibition of FNA on the photosynthetic activity and growth of microalgae, and improve the ammonia nitrogen removal efficiency and operation stability of the algal-bacterial system.

[0005] To achieve the above technical purposes, the technical scheme adopted by the present application is as follows: A method for reducing the toxic inhibition of free nitrous acid on microalgae, the method comprising: exogenously adding alpha-ketoglutarate in a microalgae-nitrifying bacteria co-culture system; the microalgae-nitrifying bacteria co-culture system is composed of wastewater, microalgae and sludge; Further, the final concentration of the alpha-ketoglutarate in the co-culture system is 0.5-1.0 mM.

[0006] Further, the microalgae include yellow silk algae, chlorella and grate algae.

[0007] Specifically, the microalgae refer to microalgae in the logarithmic phase.

[0008] Further, the microalgae and the sludge are in a dry weight, and the usage relationship of the microalgae to the wastewater is 0.2 g / L, and the usage relationship of the sludge to the wastewater is 2 g / L.

[0009] Further, the wastewater includes agricultural ammonia-nitrogen wastewater, and the ammonia-nitrogen content is >100 mg / L.

[0010] Further, the sludge contains nitrifying bacteria, and the ammonia-nitrogen removal rate to the wastewater can be continuously and stably kept above 85%.

[0011] Further, the pH value of the microalgae-nitrifying bacteria co-culture system is 6.0-7.5.

[0012] The method disclosed in the application is applied to wastewater treatment, and is specifically used for reducing the toxic inhibition of FNA on microalgae, maintaining the biological activity of microalgae in wastewater, and effectively removing ammonia-nitrogen in wastewater, with a removal rate of above 90%.

[0013] Compared with the prior art, the application has the following beneficial effects: (1) The method for reducing the toxic inhibition of FNA on microalgae disclosed in the application can effectively regulate the carbon-nitrogen metabolism in microalgae cells and enhance the physiological tolerance of the microalgae to FNA stress by exogenously adding a small amount of alpha-ketoglutarate; specifically, the method significantly reduces the damage degree of FNA to the photosynthetic growth of microalgae and the destruction of the algal-bacterial symbiotic system, and is embodied in the following aspects: the photosynthetic efficiency of microalgae (the maximum photochemical efficiency of photosystem II of microalgae photosynthetic activity Fv / Fm) is stably kept at 0.5-0.6, and the biomass accumulation of microalgae (the biomass of microalgae in the treatment group with the addition of alpha-ketoglutarate (0.8 g dry weight / L) is 2.7 times that of the control group without the addition of alpha-ketoglutarate (0.3 g dry weight / L)) and the ammonia-nitrogen removal (the ammonia-nitrogen removal rate of the treatment group with the addition of alpha-ketoglutarate (88-92.3%) is 1.5-4.0 times that of the control group without the addition of alpha-ketoglutarate (22-60.8%)).

[0014] (2) The method for reducing the toxicity of free nitrite to microalgae provided by the present invention is simple to operate and low in cost. It is applicable to actual wastewater treatment scenarios and helps to improve the long-term operational stability of algae-bacteria symbiotic systems. Attached Figure Description

[0015] Figure 1 The microalgal biomass in the algae-bacteria co-culture system of the control and treatment groups in Example 1 is ( Figure 1 A), photosynthetic activity Fv / Fm ( Figure 1 B) and the removal rate of ammonia nitrogen in wastewater ( Figure 1 The changes in C).

[0016] Figure 2 The concentration of FNA in the algae-bacteria co-culture system of the control and treatment groups in Example 2 is ( Figure 2 A) Ammonia nitrogen concentration ( Figure 2 B) Nitrite nitrogen concentration ( Figure 2 C) and chlorophyll concentration ( Figure 2 The changes in D).

[0017] Figure 3 To implement the ammonia nitrogen concentration in the algae-bacteria co-culture system of the control and treatment groups in Case 3 ( Figure 3 A) Nitrate nitrogen and nitrite nitrogen concentrations ( Figure 3 B) Chlorophyll content ( Figure 3 C) and ammonia nitrogen removal distribution ( Figure 3 The changes in D). Detailed Implementation

[0018] The technical solutions of the present invention will be further described below through specific embodiments. It should be understood that these embodiments are preferred solutions of the present invention and are intended to aid in understanding the technical content of the present invention, rather than limiting the scope of protection of the present invention. Without departing from the basic concept of the present invention, various substitutions, equivalent improvements, or combinations can be made by those skilled in the art, and all such substitutions, equivalent improvements, or combinations should be considered to fall within the scope of protection of the present invention.

[0019] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0020] The artificial wastewater preparation method in this invention refers to Integrated Partial Nitrification and Tribonema minuscultivation for cost-effective ammonia recovery and lipidproduction from slaughterhouse wastewater(Chemical Engineering Journal, 492,152199.)。

[0021] The culture medium used in the embodiments of the present application is a commonly used culture medium in the art, which is commercially available. The algae species (including yellow silk algae, Chlorella and Scenedesmus) used are conventional commercially available products (obtained from the Chinese Academy of Sciences Freshwater Algae Culture Collection) and are public materials.

[0022] The activated sludge was collected from the aerobic tank of the breeding wastewater of a pig farm in Zhenjiang, Jiangsu Province. The ammonia nitrogen removal rate of the activated sludge after 12 h of aeration was determined. If the removal rate was greater than 85%, the activated sludge was determined to be rich in nitrifying bacteria. If the removal rate was less than 85%, the activated sludge was domesticated to obtain activated sludge with increased nitrifying bacteria content. The domestication steps were as follows: the ammonia nitrogen concentration in the influent was gradually increased from 30 mg / L to the target ammonia nitrogen concentration in the breeding wastewater until the ammonia nitrogen removal rate of the activated sludge in the breeding wastewater was continuously and stably above 85%, i.e., the activated sludge rich in nitrifying bacteria was obtained.

[0023] Example 1: A yellow silk algae (Tribonema sp.) -nitrifying bacteria co-culture system was established in a laboratory-scale conical glass photobioreactor (model: height x inner diameter: 22 cm x 6 cm, total volume: 0.8 L; source: Taixing Heliang Glass Instrument Trading Co., Ltd.). The method was as follows: yellow silk algae (Tribonema sp.) cultured to the logarithmic phase in BG11 medium were collected, centrifuged, and the algal bodies were collected for use. Tribonema minus Tribonema minus The activated sludge rich in nitrifying bacteria was centrifuged to obtain the precipitate, which was inoculated into artificial wastewater together with the microalgal algal bodies (yellow silk algae in this embodiment) to construct a yellow silk algae (Tribonema sp.) -nitrifying bacteria co-culture system.

[0024] The activated sludge rich in nitrifying bacteria was centrifuged to obtain the precipitate, which was inoculated into artificial wastewater together with the microalgal algal bodies (yellow silk algae in this embodiment) to construct a yellow silk algae (Tribonema sp.) -nitrifying bacteria co-culture system. T. minus ​A nitrifying bacteria co-culture system was used, with an initial microalgae inoculum of 0.2 g dry weight / L and an initial activated sludge inoculum of 2 g dry weight / L in the wastewater. The specific composition of the artificial wastewater is shown in Table 1. Co-culture was conducted under 24-hour continuous light (intensity 12000 Lux) and 25-27 °C room temperature conditions. Simultaneously, air was continuously pumped into the water for aeration and mixing to maintain a dissolved oxygen concentration of 1-2 mg / L. The hydraulic retention time was set at 8 hours, and the pH was controlled at 6.5. During the operation of the algae-bacterial co-culture system, two groups were studied: α-ketoglutaric acid was added to the treatment group every 5 days to achieve a final concentration of 0.5 mM in the algae-bacterial co-culture system, while an equal volume of distilled water was added to the control group. Samples were periodically taken from both the treatment and control groups to monitor microalgae photosynthetic activity (Fv / Fm), microalgae chlorophyll content, and ammonia nitrogen removal rate.

[0025] Table 1. Composition of artificial wastewater

[0026] The results are as follows Figure 1 The results showed that, compared with the control group, the microalgae in the treatment group maintained normal growth, and their Fv / Fm values ​​remained within the normal range of 0.5-0.6, while the Fv / Fm values ​​in the control group continued to decrease to around 0.1. Figure 1 B). The Fv / Fm value is an important indicator for observing the stress of FNA on microalgal photosynthetic activity. This phenomenon indicates that the treatment group with α-ketoglutarate added to the algae-bacteria co-culture system did not experience microalgal death and collapse due to FNA accumulation, and the final microalgal biomass of the treatment group reached 0.8 g dry weight / L ( Figure 1 A), which is 2.7 times that of the control group, and the ammonia nitrogen removal rate has been maintained above 80% ( Figure 1 (C) indicates that, compared to the control group, the treatment group with added α-ketoglutarate can effectively maintain the biological activity of the algae-bacteria co-culture system and its removal effect on ammonia nitrogen in wastewater, achieving unexpected and significant results. Example 2:

[0027] To verify the effectiveness of the method for reducing the toxicity of free nitrite to microalgae described in this invention, practical application tests were conducted in this embodiment. Furthermore, to verify its versatility across different microalgae systems, the *Phyllostachys edulis*-nitrifying bacteria co-culture system was replaced with *Chlorella vulgaris*. Chlorella sp.) -- Nitrifying bacteria co-culture system.

[0028] Activated sludge rich in nitrifying bacteria and aquaculture wastewater (ammonia nitrogen 114.5 mg / L, COD 97.6 mg / L, nitrate nitrogen 3.3 mg N / L, total phosphorus 14.8 mg / L, nitrite nitrogen 2 mg / L) were collected from the aerobic pond of a pig farm in Zhenjiang City, Jiangsu Province, and used as influent. Single-celled Chlorella (…) Chlorella sp The algae were cultured in BG11 medium to the logarithmic growth phase, and the algal cells were collected by centrifugation. Then, *Chlorella vulgaris* algal cells and activated sludge rich in nitrifying bacteria (tested to achieve a consistently high ammonia nitrogen removal rate of over 85% in aquaculture wastewater) were inoculated into the aquaculture wastewater at initial inoculum levels of 0.2 g / L dry weight and 2 g / L dry weight, respectively, to obtain an algae-bacterial co-culture system. A raceway tank (length × width × height: 2.5 m × 1.1 m × 55 cm; stirring speed 75 rpm) was used to simulate the actual wastewater treatment process under outdoor room temperature conditions.

[0029] When the FNA concentration was ≥40 μg N / L, the algae-bacterial co-culture system was divided into two groups: α-ketoglutarate was added to the treatment group to make its final concentration in the co-culture system reach 1.0 mM, and the same volume of distilled water was added to the control group. The FNA concentration, chlorophyll content, ammonia nitrogen and nitrite nitrogen concentration in the algae-bacterial co-culture system were measured every 12 h.

[0030] The results are as follows Figure 2 As shown, the trends in FNA concentration changes were consistent between the treatment group and the control group. Figure 2 (A) However, compared to the control group, the addition of α-ketoglutarate in the treatment group was able to maintain the growth of microalgae in the algae-bacteria co-culture system, and the ammonia nitrogen removal rate reached 91.2%, which was 43.8% higher than that of the control group. Figure 2 B), under the action of microalgae photosynthetic oxygen production, the nitrification rate of ammonia nitrogen reached 93.3%, which was 42.3% higher than that of the control group. Figure 2 C), microalgal growth was not inhibited, and the chlorophyll content reached 9.5 mg / L ( Figure 2 (D) indicates that the algae co-culture system in the treatment group did not experience microalgae death and collapse due to FNA accumulation. Example 3:

[0031] To verify the effectiveness of the method for reducing the toxicity of FNA to microalgae described in this invention, practical application tests were conducted in this embodiment. Furthermore, to verify its versatility across different microalgae systems, the *Phyllostachys edulis*-nitrifying bacteria co-culture system was replaced with *Scenedesmus*. Scenedesmus sp.) -- Nitrifying bacteria co-culture system.

[0032] Activated sludge rich in nitrifying bacteria (same as in Example 1, tested to consistently achieve an ammonia nitrogen removal rate of over 85% in the livestock wastewater; if not, acclimatization was performed) and livestock wastewater (ammonia nitrogen content 221 mg / L, COD 247 mg / L, nitrate nitrogen 3.3 mg N / L, total phosphorus content 10.8 mg / L, nitrite nitrogen content 1 mg / L, pH 7.5) were collected from the aerobic pond of a pig farm in Zhenjiang City, Jiangsu Province, and used as the influent for the algae-bacteria co-culture system. Single-celled Scenedesmus (… Scenedesmus The algae (sp.) were used as the inoculum and cultured in BG11 medium to the logarithmic growth phase. The algal cells were then collected by centrifugation. Single-celled Scenedesmus algae and activated sludge rich in nitrifying bacteria were then inoculated into aquaculture wastewater at initial inoculum levels of 0.2 g / L dry weight and 2 g / L dry weight, respectively, to obtain an algae-bacterial co-culture system. A raceway tank (length × width × height: 2.5m × 1.1m × 55cm; stirring speed 75 rpm) was used to simulate the actual wastewater treatment process under outdoor room temperature conditions.

[0033] When the FNA concentration was ≥40 μg N / L, the algae-bacterial co-culture system was divided into two groups: α-ketoglutarate was added to the treatment group to make its final concentration in the co-culture system reach 0.75 mM, and the same volume of distilled water was added to the control group. The chlorophyll content, ammonia nitrogen, nitrite nitrogen and nitrate nitrogen concentration in the algae-bacterial co-culture system were measured every 12 h.

[0034] The results are as follows Figure 3 As shown, compared to the control group, the addition of α-ketoglutarate in the treatment group was able to maintain the growth of microalgae in the algae-bacteria co-culture system, with a chlorophyll content reaching 16.8 mg / L, while the control group showed a significant decrease in chlorophyll content and microalgae death. Figure 3 C); The treatment group achieved ammonia nitrogen removal rate of 92.3%, which was 31.5% higher than the control group. Figure 3 In group A, the nitrification rate of ammonia nitrogen reached 56.1% under the action of microalgal photosynthetic oxygen production, and the concentration of low-toxicity nitrate nitrogen reached 74.9 mg / L, which was 30.2 mg / L higher than that of the control group. Figure 3 B). Meanwhile, in the treatment group, 30.1% of the ammonia nitrogen was removed by microalgae absorption and conversion, while in the control group, only 8.3% of the ammonia nitrogen was removed by microalgae absorption. Figure 3 (D) This indicates that the algae-bacteria co-culture system in the treatment group did not experience microalgae death and collapse due to FNA accumulation, and that the biotransformation efficiency of microalgae for ammonia nitrogen pollutants in wastewater was enhanced.

[0035] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for reducing the inhibitory toxicity of free nitrite to microalgae, characterized in that, The method includes: exogenously adding α-ketoglutarate to a microalgae-nitrifying bacteria co-culture system; the microalgae-nitrifying bacteria co-culture system is composed of wastewater, microalgae and sludge.

2. The method for reducing the toxicity of free nitrite to microalgae according to claim 1, characterized in that, The final concentration of α-ketoglutarate in the co-culture system is 0.5-1.0 mM.

3. The method for reducing the inhibitory effect of free nitrite on microalgae toxicity according to claim 1, characterized in that, The microalgae include *Hydrocotyle spp.*, *Chlorella vulgaris*, and *Scenedesmus*.

4. The method for reducing the toxicity of free nitrite to microalgae according to claim 3, characterized in that, The microalgae referred to are those in the logarithmic phase.

5. The method for reducing the inhibitory effect of free nitrite on microalgae toxicity according to claim 1, characterized in that, On a dry weight basis, the dosage of microalgae to wastewater is 0.2 g / L, and the dosage of sludge to wastewater is 2 g / L.

6. The method for reducing the inhibitory effect of free nitrite on microalgae toxicity according to claim 5, characterized in that, The wastewater includes agricultural ammonia nitrogen wastewater with an ammonia nitrogen content >100 mg / L.

7. The method for reducing the toxicity of free nitrite to microalgae according to claim 5, characterized in that, The sludge contains nitrifying bacteria and can continuously and stably remove ammonia nitrogen from wastewater at a rate of over 85%.

8. The method according to claim 1, characterized in that, The pH value of the microalgae-nitrifying bacteria co-culture system is 6.0-7.

5.

9. The application of the method according to any one of claims 1-8 in wastewater treatment.

10. The application according to claim 9, characterized in that, The application is used to reduce the toxicity of free nitrite to microalgae, maintain the biological activity of microalgae in wastewater, and effectively remove ammonia nitrogen from wastewater with a removal rate of over 90%.

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