A strain of Achromobacterium and a method for synergistic treatment of aquaculture wastewater with microalgae

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Achromobacterium* and a method for synergistically treating aquaculture wastewater with microalgae. The specific technical solution is as follows: a novel *Achromobacterium* strain, with accession number CGMCC No. 28350. The *Achromobacterium* is used in conjunction with *Protozoa* for wastewater treatment. This invention provides a synergistic algae-bacteria treatment system. *Protozoa* absorbs CO2 to synthesize organic matter, continuously providing a carbon source for aerobic denitrifying bacteria, improving their activity, and promoting the denitrification efficiency of the bio-enhanced treatment system. The oxygen released by the algae supplements the respiratory metabolic needs of the aerobic denitrifying bacteria, reducing the aeration energy consumption of the treatment system. Combining aerobic denitrifying bacteria with microalgae into a bio-enhanced treatment system solves the problem of slow nitrate absorption rate in RAS wastewater by microalgae and alleviates the problem of insufficient carbon source for aerobic denitrifying bacteria alone. Simultaneously, the aerobic denitrifying bacteria can also improve the amino acid composition of *Protozoa*, increase amino acid production, and enhance the feed value of *Protozoa*.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of colorless bacillus and a method for synergistic treatment of aquaculture wastewater with microalgae. Background Technology

[0002] Recirculating aquaculture systems (RAS) typically include physical or biological treatment units. Physical filtration removes fish waste and feed residue from the circulating water, while microbial nitrification converts toxic ammonia and nitrite nitrogen into nitrate nitrogen, thus detoxifying the wastewater. Consequently, RAS effluent generally exhibits high levels of nitrate nitrogen and total phosphorus. In RAS systems lacking denitrification units or with inadequate denitrification, nitrate nitrogen concentrations can reach 400–500 mg / L. Existing research indicates that nitrate nitrogen may be an endocrine disruptor, affecting the growth and development of aquatic organisms. Furthermore, high levels of nitrate nitrogen not only lead to eutrophication but also further harm the health of aquatic organisms and waste nutrients.

[0003] Microalgae possess advantages such as short growth cycle, rapid growth rate, high photosynthetic efficiency, and wide adaptability, enabling them to grow in various types of wastewater and making them an effective method for wastewater treatment. Simultaneously, microalgae are rich in proteins, polysaccharides, lipids, pigments, vitamins, and unsaturated fatty acids, making them widely applicable in animal feed, such as aquaculture feed. Compared to traditional fish feed, microalgae feed contains a greater variety of essential amino acids and unsaturated fatty acids, making it an ideal protein source for aquaculture feed. Therefore, using microalgae in aquaculture wastewater treatment offers at least the following advantages: 1. It can remove pollutants from wastewater efficiently and with low carbon footprint, and effectively recover nitrogen and phosphorus elements; 2. Microalgae can serve as a protein source for aquatic animals, reducing dependence on traditional animal feeds (such as fishmeal) and promoting the sustainable development of the aquaculture industry; 3. Microalgae produce oxygen during photosynthesis, increasing the oxygen content of the water, improving the aquatic ecological environment, and enhancing aquaculture yield and quality.

[0004] However, nitrogen in RAS water mainly exists in the form of nitrate nitrogen, which is not a readily available nitrogen source for microalgae. Nitrate nitrogen usually needs to be converted into ammonia nitrogen within microalgal cells before it can be assimilated by the cells. Therefore, the assimilation rate of nitrate nitrogen by microalgae is relatively slow, which greatly limits the treatment efficiency of microalgae in RAS effluent.

[0005] Aerobic denitrifying bacteria are a type of bacteria that can perform denitrification under aerobic conditions. Compared to traditional denitrifying bacteria, which require anaerobic or hypoxic conditions to function, aerobic denitrifying bacteria are more suitable for the high dissolved oxygen (DO) environment of aquaculture. However, the carbon-to-nitrogen ratio in aquaculture water is usually below 5, and the lack of carbon sources greatly limits the nitrogen removal performance of aerobic denitrifying bacteria.

[0006] Therefore, if a microalgae-bacteria system can be established, it is expected to improve the treatment effect of sewage by microalgae and efficiently remove pollutants from sewage. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of colorless bacillus and a method for synergistic treatment of aquaculture wastewater with microalgae.

[0008] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: a new strain of Achromobacter xylosoxidans subsp. denitrificans AD-Z, which was deposited on September 4, 2023, at the China General Microbiological Culture Collection Center, with the accession number CGMCC No. 28350.

[0009] Accordingly, the application of the achromobacterium in wastewater treatment.

[0010] Preferably, the colorless bacillus is used in combination with protozoan algae for wastewater treatment.

[0011] Preferably, it is used in the removal of ammonia nitrogen, nitrate nitrogen, phosphorus and COD from wastewater.

[0012] Preferably, the protothecoides is Auxenochlorella protothecoides H34#, which was deposited on November 2, 2022 at the China General Microbiological Culture Collection Center with accession number CGMCC No. 45371.

[0013] Preferably, the application conditions are: controlling the ambient temperature to be 20℃~30℃; controlling the light intensity to be 5000lx; and controlling the light-dark cycle to be 12h:12h.

[0014] Accordingly, the colorless bacillus is used in increasing the amino acid content of Protozoa; and / or increasing the proportion of essential amino acids in Protozoa.

[0015] Preferably, the protothecoides is Auxenochlorella protothecoides H34#, which was deposited on November 2, 2022 at the China General Microbiological Culture Collection Center with accession number CGMCC No. 45371.

[0016] Preferably, the application method includes: inoculating the colorless bacillus and the protozoan into the same environment for co-culture.

[0017] Correspondingly, a strain of *Auxenochlorella protothecoides* H34# was deposited on November 2, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45371.

[0018] This invention offers the following advantages: To address the current problem of ineffective nitrogen and phosphorus recovery in reclaimed air (RAS) water, this invention provides a synergistic algae-bacteria treatment system, specifically comprising *Protozoa* H34# and aerobic denitrifying bacteria AD-Z. During photosynthesis, *Protozoa* H34# absorbs CO2 to synthesize organic matter, continuously providing a carbon source for aerobic denitrifying bacteria AD-Z, thus enhancing AD-Z's activity and promoting the denitrification efficiency of the bioaugmented treatment system. Furthermore, the oxygen released by the algae replenishes the respiratory metabolic needs of the aerobic denitrifying bacteria, reducing the aeration energy consumption of the treatment system. Therefore, combining aerobic denitrifying bacteria with microalgae in a bioaugmented treatment system for RAS effluent treatment not only solves the problem of slow nitrate and nitrogen absorption rates by microalgae in RAS effluent but also alleviates the carbon source deficiency of aerobic denitrifying bacteria alone. Simultaneously, aerobic denitrifying bacteria AD-Z can improve the amino acid composition of *Protozoa* H34#, increasing amino acid production and effectively enhancing the feed value of *Protozoa* H34#. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of Achromobacterium AD-Z.

[0020] Figure 2 A schematic diagram showing the changes in dry weight of Protozoa H34# under different treatment conditions;

[0021] Figure 3 A schematic diagram showing the changes in photosynthetic pigments of Protozoa H34# under different treatment conditions;

[0022] Figure 4 A schematic diagram showing the changes in ammonia nitrogen in RAS effluent under different treatment conditions;

[0023] Figure 5 A schematic diagram showing the changes in nitrate nitrogen in RAS effluent under different treatment conditions;

[0024] Figure 6 A schematic diagram showing the changes in total nitrogen in RAS effluent under different treatment conditions;

[0025] Figure 7 A schematic diagram showing the changes in total phosphorus in RAS effluent under different treatment conditions;

[0026] Figure 8 A schematic diagram showing the changes in photosynthetic pigments of Protozoa H34# under different algal-bacterial inoculation ratios;

[0027] Figure 9 A schematic diagram showing the changes in nitrate nitrogen in RAS effluent under different algal and bacterial inoculation ratios;

[0028] Figure 10 A schematic diagram showing the changes in total nitrogen in RAS effluent under different algal and bacterial inoculation ratios;

[0029] Figure 11 A schematic diagram showing the changes in total phosphorus in RAS effluent under different algal and bacterial inoculation ratios;

[0030] Figure 12 A schematic diagram showing the changes in dry weight of Protozoa H34# under different culture conditions;

[0031] Figure 13 A schematic diagram showing the changes in photosynthetic pigments of Protozoa H34# under different culture conditions;

[0032] Figure 14 A schematic diagram showing the changes in nitrate and nitrogen in RAS tailwater under different culture conditions;

[0033] Figure 15 A schematic diagram showing the changes in total nitrogen in RAS tailwater under different culture conditions;

[0034] Figure 16 A schematic diagram showing the changes in total phosphorus in RAS tailwater under different culture conditions;

[0035] Figure 17 A schematic diagram showing the changes in total amino acid content of Protozoa H34# under different culture conditions. Detailed Implementation

[0036] This invention provides a novel microalga, *Auxenochlorella protothecoides* H34#, abbreviated as *Auxenochlorella protothecoides* or *Auxenochlorella protothecoides* H34#, obtained by screening *Auxenochlorella protothecoides* CK-2# after heavy ion irradiation. *Auxenochlorella protothecoides* H34# was deposited on November 2, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 45371. The ITS sequence of *Auxenochlorella protothecoides* H34# is shown in SEQ ID NO:1. *Auxenochlorella protothecoides* H34# exhibits rapid growth, genetic stability, and adaptability to environments with high ammonia nitrogen, high (nitrite) nitrogen, high phosphorus, and high COD. It can effectively remove and degrade nitrogen and phosphorus in wastewater, such as RAS effluent, thereby reducing wastewater COD.

[0037] This invention also provides a novel *Achromobacter xylosoxidans* subsp. *denitrificans* AD-Z, abbreviated as *Achromobacter* or *Achromobacter* AD-Z. *Achromobacter* AD-Z was deposited on September 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 28350. The 16S rDNA sequence of *Achromobacter* AD-Z is shown in SEQ ID NO:2. *Achromobacter* AD-Z can adapt to environments with high ammonia nitrogen, high (nitrite) nitrogen, high phosphorus, and high COD, and can effectively remove and degrade nitrogen and phosphorus in wastewater, such as RAS effluent, reducing wastewater COD. It can also be used in combination with the *Protozoa* H34# for wastewater treatment to improve wastewater treatment efficiency and effectiveness. Meanwhile, Achromobacterium AD-Z can also promote the growth of Protozoa H34#, increase the amino acid content of Protozoa H34#, and improve the amino acid composition of Protozoa H34#.

[0038] The present invention also provides a wastewater treatment method based on the aforementioned Protozoa H34# and the aforementioned Achromobacterium AD-Z, specifically by inoculating the Protozoa alone or in combination with Achromobacterium AD-Z into the wastewater to be treated (Achromobacterium AD-Z can also be inoculated into the wastewater to be treated alone).

[0039] The preferred method is as follows: after inoculating the wastewater to be treated with Protozoa and / or Achromobacterium, the ambient temperature is controlled at 20℃~30℃, preferably 25℃; the light intensity is controlled at 5000lx, and the light-dark cycle is 12h:12h.

[0040] The present invention also provides a method for producing algal protein / algal amino acids and improving the amino acid composition of algal protein, specifically by co-culturing the Protozoa H34# and the Achromobacterium AD-Z.

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0042] Example 1: Obtaining Protocrustae and Demonstrating Their Wastewater Treatment Effect

[0043] 1. Obtaining Protocellus.

[0044] Auxenochlorella protothecoides CK-2# was selected and subjected to heavy ion irradiation. After multiple screenings considering growth, genetic stability, and nitrogen and phosphorus tolerance, a mutant Auxenochlorella protothecoides H34# with fast growth and genetic stability was obtained. The ITS sequence of Auxenochlorella protothecoides H34# is shown in SEQ ID NO:1, confirming its identity as Auxenochlorella protothecoides. The physiological and biochemical characteristics of Auxenochlorella protothecoides H34# include: tolerance to high concentrations of ammonia and nitrate nitrogen; high content of algal cell protein and lipids; tolerable temperature range: 10℃~35℃; optimal growth temperature: 25℃~30℃; tolerable pH range: 5~11; optimal growth pH: 8~10; suitable light conditions: 6000lx~8000lx, preferably 5000lx, with a light-dark cycle of 12h:12h; and suitable growth medium: BG11 medium. The *Protocolospora* H34# was deposited on November 2, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 45371.

[0045] 2. The effect of using Protozoa alone to treat wastewater.

[0046] The *Protozoa* H34# was inoculated at an OD value. 680 =0.2 mg / L was inoculated into 200 mL of aquaculture wastewater and cultured in shake flasks in a light incubator. The light intensity was 5000 lx, the light-dark cycle was 12 h:12 h, and the temperature was 25 ± 1 °C. Treatment continued for 10 days, with water quality indicators and the growth status of *Protoplastrum hyaline* H34# measured every two days. A blank control group was set up (no *Protoplastrum hyaline* H34# inoculated, all other treatments were the same); a positive control group was set up (inoculated with an equal amount of *Protoplastrum hyaline* CK-2#, all other treatments were the same). Each group had three replicates, and the results were averaged. The initial total nitrogen in the aquaculture wastewater was 31.57 mg / L; the initial total phosphorus was 2.07 mg / L; the initial ammonia nitrogen content was 4.48 mg / L; the initial nitrite nitrogen content was 0.46 mg / L; the initial nitrate nitrogen content was 28.08 mg / L; and the initial COD was 51.56 mg / L.

[0047] In wastewater quality testing, ammonia nitrogen was determined using Nessler's reagent spectrophotometry (HJ 535-2009), nitrite nitrogen using N-(1-naphthyl)-1,2-ethylenediamine dihydrochloride spectrophotometry (GB 7493-1987), nitrate nitrogen using ultraviolet spectrophotometry (HJ / T 346-2007), total nitrogen using alkaline potassium persulfate digestion ultraviolet spectrophotometry (GB 11894-1989), total phosphorus using ammonium molybdate spectrophotometry (GB 11893-89), and chemical oxygen demand (COD) using rapid digestion spectrophotometry (HJ / T 399-2007). Subsequent tests for the same indicators followed the same methods and will not be repeated here. The results are shown in Table 1.

[0048] Table 1 Comparison of Wastewater Treatment Results Using Protozoa

[0049]

[0050] The results showed that compared with the recalcitrant organic matter in industrial wastewater, the organic matter in aquaculture effluent was relatively easy to degrade and could be utilized by microorganisms in the natural environment. Because naturally occurring organic-degrading microorganisms existed in the raw water, the COD in the blank control group decreased significantly. In the *Protozoa* group, in addition to the natural decomposition of COD by microorganisms, the microalgae also secreted organic matter into the water during their growth, increasing the COD; therefore, the COD was higher than that of the blank control group. *Protozoa* H34# showed good adaptability to aquaculture wastewater, with rapid pollutant removal rates and high biomass accumulation.

[0051] Example 2: Screening of aerobic denitrifying bacteria and demonstration of their effect on treating effluent

[0052] 1. Microbial Screening. Water samples were taken from the overlying water of an open-air fish farm in Meishan City, Sichuan Province. 10 mL of the original water sample was inoculated into 90 mL of heteroaerobic nitrification medium (HNM medium) and cultured at 25℃ and 140 rpm for 3 days. This was repeated three times to enrich the dominant bacterial strains. The bacterial suspension was then serially diluted with physiological saline and evenly spread onto HNM plates, which were then incubated at 25℃. After colony formation, single colonies of different morphologies were picked and further purified until single colonies were obtained. The purified strains were inoculated onto BTB solid medium (1 mL of 1% bromothymol blue ethanol solution was added to 1 L of DM medium, and 15 g of agar was added for sterilization to obtain BTB solid medium). The surrounding area of ​​the plate turned blue. Strains that turned blue exhibited aerobic denitrification function.

[0053] After screening and purification, strain AD-Z with aerobic denitrification function was obtained. Strain AD-Z was inoculated into aerobic denitrification medium (DM) and cultured at 25℃ and 140 rpm. The colony morphology of AD-Z is shown in the figure below. Figure 1 As shown. Physiological and biochemical characteristics include: Gram-negative; tolerable temperature: 15–45℃; optimal growth temperature: 25–35℃; tolerable pH: 5–10; optimal growth pH: 7–9; available carbon sources include sodium acetate, sodium citrate, glucose, and sodium succinate. Sequencing was used to identify the species classification of strain AD-Z. The primers for 16S rRNA gene amplification were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). DNA sequencing and splicing were performed by Shanghai Meiji Biotechnology Co., Ltd. The 16S rDNA sequence of strain AD-Z is shown in SEQ NO ID: 2. The sequenced sequences were aligned using the NCBI online BLAST program, and a phylogenetic tree was constructed using the MEGA 11 Neighbor-Joining method, with the algorithm self-checking program Bootstrap running 1000 times. AD-Z was identified as belonging to the genus Achromobacter Sp.

[0054] The achromobacterium AD-Z was deposited on September 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28350.

[0055] 2. The effect of Achromobacterium AD-Z on wastewater treatment alone.

[0056] Achromobacterium AD-Z was inoculated at an OD value. 600 =0.1 mg / L was inoculated into 200 mL of aquaculture wastewater and cultured on a shaker at 25℃ for 5 days, with water quality indicators measured every two days. A blank control group was set up, without inoculation of *Achromobacterium AD-Z*, with all other treatments the same; a positive control group was set up, inoculated with an equal amount of commercially available *Achromobacterium* (CGMCC No. 2964), with all other treatments the same; each group was replicated in triplicate, and the average value was taken. The initial total nitrogen content in the aquaculture wastewater was 29.45 mg / L; the initial total phosphorus content was 4.81 mg / L; the initial ammonia nitrogen content was 0.93 mg / L; the initial nitrite nitrogen content was 1.06 mg / L; the initial nitrate nitrogen content was 29.67 mg / L; and the initial COD was 25.52 mg / L. The treatment results are shown in Table 2.

[0057] Table 2 Comparison of Wastewater Treatment Results Using Achromobacterium oxysporum

[0058] Total nitrogen content on day 5 30.05 mg / L 33.32 mg / L 29.13 mg / L Total phosphorus content on day 5 4.65 mg / L 5.96 mg / L 4.55 mg / L ammonia nitrogen content on day 5 0.13 mg / L 1.12 mg / L 0.59 mg / L Nitrite content on day 5 0.06 mg / L 0.88 mg / L 1.09 mg / L Nitrogen content on day 5 25.80 mg / L 31.13 mg / L 28.60 mg / L Day 5 of COD 18.06 mg / L 49.72 mg / L 13.13 mg / L Maximum ammonia nitrogen removal rate and time to reach this value 86.02％，1d 0％，0d 36.56％，5d Maximum removal rate of nitrite and time to reach this value 94.34％，1d 16.98％，5d 0％，0d Maximum nitrogen removal rate and time to reach this value 13.04％，1d 0％，0d 3.61％，5d Maximum COD removal rate and time to reach that value 29.23％，5d 0％，0d 48.55％，5d

[0059] Because the initial COD content in the raw wastewater is low, the carbon source cannot meet the continuous growth requirements of *Achromobacterium* AD-Z, resulting in a low pollutant removal rate for AD-Z alone. Commercially available *Achromobacterium* cannot utilize COD, leading to nutrient deficiency and bacterial death, which in turn causes an increase in pollutants in the water. AD-Z may also experience bacterial death in its later growth stages, leading to an increase in total nitrogen content.

[0060] Example 3: Demonstration of the effect of combined algae and bacteria treatment of wastewater

[0061] 1. Feasibility verification of co-treatment of wastewater by algae and bacteria.

[0062] Four experimental groups were set up: H34#, H34# + activated sludge, H34# + aerobic denitrifying bacteria, and nitrifying bacteria. These were inoculated into sterilized RAS effluent according to the proportions shown in Table 3. The groups were then cultured in shake flasks under a light incubator with a light intensity of 5000 lx, a light-dark cycle of 12 h:12 h, and a temperature of 25 ± 1℃ for 10 consecutive days. Water quality indicators and algae / bacterial growth were measured every two days. A blank control group was also set up: no algae, bacteria, or activated sludge were inoculated, while all other conditions were the same. Each treatment was repeated three times, and the average value was taken. The initial pollutant concentrations in the RAS effluent were: ammonia nitrogen 0.57 mg / L, nitrite nitrogen 1.04 mg / L, nitrate nitrogen 20.03 mg / L, total nitrogen 25.94 mg / L, total phosphorus 4.61 mg / L, and COD 29.28 mg / L. Because the initial concentrations of ammonia nitrogen and nitrite nitrogen in the wastewater were low, and these concentrations remained at low levels throughout the culture period without significant changes, the main focus was on the changes in other pollutant indicators in the wastewater.

[0063] In the above experimental group, the aerobic denitrifying bacteria were a mixed bacterial system enriched with DM medium, possessing aerobic denitrification function but not purified, and containing *Achromobacterium AD-Z*. The nitrifying bacteria were a mixed bacterial system with nitrification function, obtained by placing a sample of sludge mixture from a wastewater treatment plant in a culture reactor, adding nitrification medium, adjusting the suspended solids concentration of the mixture to 3000 mg / L, pH = 8.0, and dissolved oxygen controlled at 0.5–1.5 mg / L, and replacing the nitrification medium every 12 hours. The initial ammonia nitrogen concentration was 210 mg / L, and successful acclimation was considered achieved when the ammonia nitrogen removal rate exceeded 5 mg / L / h. The mixed bacterial system contained ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB).

[0064] The growth indicators of *Protozoa* were determined as follows: the dry weight and photosynthetic pigment content of the algae were used to characterize the growth of the algal strain. The optical density (OD) was measured using a UV-Vis spectrophotometer at a wavelength of 680 nm.680 Based on the values ​​of optical density and the corresponding dry weight of algae, a standard curve was plotted according to the linear relationship between optical density and dry weight. Optical density (OD) was used as the plotting factor. 680 The dry weight of the microalgae was calculated using the ethanol extraction method (Xiaoting L, Li W, Zhai J, Wei H, Wang Q (2019) Effect of ammonium nitrogen on microalgal growth, biochemical composition and photosynthetic performance in mixotrophic cultivation. Biores Technol 273:368–376.). The contents of three chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Cx.c) in the algal strain were determined. The types and proportions of hydrolyzed amino acids in the algae were determined using a fully automated amino acid analyzer (Hitachi L8900). The changes in the growth of the protocorm are shown in the figure. Figure 2 , 3 As shown in (dry weight, photosynthetic pigments), the changes in various pollutants in wastewater are as follows: Figures 4-7 (Ammonia nitrogen, nitrate nitrogen, total nitrogen, total phosphorus) are shown.

[0065] Table 3. Algae and bacteria control for each treatment group (unit: mg / L)

[0066] Group 1 (Microalgae H34#) 30 \ \ \ Group 2 (microalgae H34# + aerobic denitrifying bacteria) 20 10 \ \ Group 3 (Microalgae H34# + Activated sludge) 20 \ 10 \ Group 4 (nitrifying bacteria) \ \ \ 30 Blank control group \ \ \ \

[0067] Although group 4 and the blank control group were not inoculated with microalgae, due to the use of OD 600 The indicator displays biomass, which can also reflect changes in bacterial biomass to some extent; nitrifying bacteria were present in group 4, while a small amount of natural bacteria or other impurities were present in the blank control group. Figure 2 This shows that the two groups are at OD 600 All of them have a small amount of absorbance. According to Figure 2 In the initial biomass (OD) 680 When the concentration was 0.1, the biomass of experimental group 1 maintained growth for the first 8 days, then decreased in the last two days; the biomass of experimental group 3 increased for the first 6 days, then stabilized for the last 4 days; while the biomass of experimental group 2 continued to grow throughout the culture period, reaching a peak of 7.4 times its initial value at the end of the culture, which was 3.41 times that of experimental group 1 and 1.72 times that of experimental group 3. This indicates that H34# single algae is difficult to stabilize in RAS effluent, and the addition of aerobic denitrifying bacteria can improve the stability of the treatment system and promote the continuous growth of microalgae biomass. Figure 3The changes in photosynthetic pigments in experimental group 1 were consistent with the changes in biomass. This is because experimental group 1 had the advantage of a high initial microalgae inoculum, resulting in the highest microalgae biomass for the first 8 days. However, the microalgae system collapsed in the last two days, and the microalgae began to die, possibly due to excessive accumulation of microalgae metabolites in the later stages of growth, leading to bioinhibition. Under algae-bacteria synergistic conditions, aerobic denitrifying bacteria could effectively utilize microalgae metabolites, alleviating this phenomenon. From day 6 onwards, the total photosynthetic pigment content in experimental group 3 remained stable and did not continue to increase; while the total photosynthetic pigment content in experimental group 2 continued to increase throughout the entire culture cycle, reaching its highest level at the end of the culture, 1.71 times that of experimental group 1 and 1.40 times that of group 3.

[0068] according to Figures 4-7 Because the initial ammonia nitrogen concentration in the wastewater was low, the ammonia nitrogen concentration remained low throughout the four experimental groups. In the later stages of the experiment, the ammonia nitrogen concentration in group 1 rose rapidly, indicating that the large-scale death of the single algae biomass led to a rapid increase in ammonia nitrogen in the water. Regarding nitrate nitrogen changes, except for group 4, the nitrate nitrogen in the other three groups decreased. The removal rate of group 2 was 100%, and that of group 3 was 97.9%, both higher than the 84.6% of group 1, indicating that the algae-bacteria system was more conducive to nitrate nitrogen removal. The changes in total nitrogen and total phosphorus were similar. Total nitrogen and total phosphorus in group 4 increased slowly during the cultivation period, while total nitrogen and total phosphorus in group 1 and total phosphorus in group 3 decreased in the first eight days and increased in the last two days. Group 2 showed the best and most stable removal effect for total nitrogen and total phosphorus, with removal rates of 94.3% and 100% respectively at the end of the cultivation period, and a removal rate of 2.45 mg·L⁻¹. -1 ·d -1 and 0.46 mg·L -1 ·d -1 .

[0069] In summary, conventional nitrifying bacteria can only convert ammonia and nitrite nitrogen in RAS effluent into nitrate nitrogen, without removing total nitrogen and total phosphorus. Microalgae, on the other hand, can remove both nitrogen and phosphorus from the water. However, single-algae systems are unstable, and microalgae may die and release nitrogen and phosphorus later on. The synergistic treatment of microalgae and aerobic denitrifying bacteria can improve the stability of the bioaugmentation system in wastewater and enhance the removal efficiency of nitrogen and phosphorus from wastewater.

[0070] 2. The impact of the ratio of algae to bacteria on wastewater treatment.

[0071] Following the methods outlined in Table 4, aerobic denitrifying bacteria AD-Z and H34# were inoculated into sterilized RAS effluent at different ratios and cultured in shake flasks under illumination. A blank control group was established, without the addition of AD-Z and microalgae, while all other treatment conditions were the same as the experimental groups. A positive control group was also established, in which AD-Z was replaced with commercially available Achromobacterium chrysogenum CGMCC No. 2964. Each group was divided into three replicates, and the results were averaged.

[0072] The initial pollutant concentrations in the RAS effluent were: ammonia nitrogen 0.43 mg / L, nitrite nitrogen 0.03 mg / L, nitrate nitrogen 28.87 mg / L, total nitrogen 29.12 mg / L, total phosphorus 4.30 mg / L, and COD 65.93 mg / L. Cultivation conditions: light intensity 5000 lx, light-dark cycle 12 h:12 h, temperature 25 ± 1℃, continuous cultivation for 10 days, with water quality indicators and growth status measured every two days. The growth was determined based on the algal optical density (OD). 680 ) and the optical density value (OD) of bacteria 600 The inoculum ratio is calculated using the ratio of bacteria to algae concentrations. Since the bacterial concentration is much higher than the algal concentration under the same light density, the bacterial ratio is multiplied by 10 before calculating the inoculum ratio. For example: Algal OD... 680 =0.1, bacterial OD 600 =0.01, recorded as 1:1.

[0073] Table 4. Comparison of Initial Vaccination Volumes

[0074] Group 1 0.1 AD-Z 0 1:0 Group 2 0.1 AD-Z 0.002 5:1 Group 3 0.1 AD-Z 0.003 3:1 Group 4 0.1 AD-Z 0.01 1:1 Group 5 0.1 AD-Z 0.03 1:3 Group 6 0.1 AD-Z 0.05 1:5 Blank control group \ \ \ Positive control group 0.1 Commercially available Achromobacterium 0.03 1:3

[0075] Changes in photosynthetic pigments in each group are as follows: Figure 8 As shown, the changes in pollutants in wastewater are as follows: Figure 9 , 10 As shown in Figure 11. The results showed that, in terms of microalgae growth, the total photosynthetic pigments in the 1:5 and 1:3 ratios were significantly higher than those in other ratios, with the 1:5 ratio having the highest total photosynthetic pigment at 13.24 mg / L, which was 1.13 times that of the 1:0 algae-to-bacteria ratio. Regarding nitrate removal, the 1:3 and 3:1 ratios were more effective than other combinations, with a removal rate of 100%. Regarding total nitrogen removal, the 1:3, 5:1, and 3:1 ratios were more effective than other combinations, with the 1:3 ratio showing the best effect at 83.24%. Regarding total phosphorus removal, the 1:3 and 3:1 ratios were more effective than other combinations, with the 1:3 ratio showing the best effect at 92.7%. In summary, the 1:3 ratio was the most effective, with removal rates of 100%, 83.24%, and 92.7% for nitrate, total nitrogen, and total phosphorus, respectively, and a removal rate of 3.51 mg / L. -1 ·d -1 2.84 mg·L -1 ·d-1 and 0.48 mg·L -1 ·d -1 The removal rates were 26.4%, 19.8%, and 17.5% higher than those of single algae (Group 1), respectively. Replacing Achromobacterium AD-Z with commercially available Achromobacterium did not significantly improve microalgal biomass or pollutant removal efficiency compared to Group 1, indicating that not all Achromobacterium species can achieve a good synergistic effect with microalgae.

[0076] 3. Demonstration of wastewater treatment effect under optimal treatment conditions.

[0077] After obtaining the optimal initial inoculation ratio of algae and bacteria, the pollutant removal process of H34# and H34#+AD-Z on RAS effluent was demonstrated using an optimal inoculation ratio of 1:3. The initial pollutant concentrations in the RAS effluent were: ammonia nitrogen 0.93 mg / L, nitrite nitrogen 1.06 mg / L, nitrate nitrogen 29.67 mg / L, total nitrogen 30.45 mg / L, total phosphorus 4.81 mg / L, and COD 25.52 mg / L. Shake-flask culture was conducted in a light-shaking incubator with a light intensity of 5000 lx, a light-dark cycle of 12 h:12 h, and a temperature of 25 ± 1℃ for 10 consecutive days. Water quality indicators and growth status were measured periodically.

[0078] Changes in microalgal biomass as follows Figure 12 As shown, the changes in photosynthetic pigments are as follows: Figure 13 As shown in the figure. The results showed that on the last day, the biomass of the H34#+AD-Z group reached its highest level, which was 1.09 times that of the single algae group. The total photosynthetic pigments of the algae and bacteria in each group did not differ significantly and remained basically the same on the last day, indicating that the biomass of microalgae in the algae-bacteria combination was not significantly different. The reason for the difference in overall biomass was the increase in the biomass of aerobic denitrifying bacteria. This indicates that the biomass of microalgae and aerobic denitrifying bacteria AD-Z increased simultaneously, forming a positive symbiotic relationship between the algae and bacteria, achieving a synergistic growth effect. The changes in pollutants in the wastewater are shown in the figure. Figure 14 , 15 As shown in Figure 16, the efficiency of the algae-bacteria group in treating pollutants was significantly higher than that of the single algae treatment, which also proves that there is a synergistic effect between algae and bacteria.

[0079] 4. Changes in amino acid composition in H34#.

[0080] After the cultivation in step 3 of this embodiment, H34# (Protozoa 1) from the H34#+AD-Z group and H34# (Protozoa 2) from the H34# group were collected. Simultaneously, under the following conditions: light intensity of 5000 lx and a light-dark cycle of 12 h:12 h, H34# Protozoa were cultured in BG11 medium for 10 days, and then H34# (Protozoa 3) was collected. The amino acid composition of Protozoa 1, 2, and 3 was determined to ascertain the feed potential of Protozoa obtained by different methods.

[0081] The results are as follows Figure 17 As shown in Table 5.

[0082] Table 5 Comparison of amino acid composition in various Protochalcogenides

[0083]

[0084]

[0085] The results showed that the total amino acid content of *Protozoa 1* was 37.84%, significantly higher than that of *Protozoa 2*, and very close to that of *Protozoa 3* cultured in the ideal growth environment BG11. This indicates that *Achromobacterium AD-Z* can significantly increase the amino acid content in *Protozoa H34#*. Furthermore, according to the ideal protein reference model proposed by WHO / FAO, the essential amino acid / total amino acid ratio (EAA / TAA) should reach 40%, and the essential amino acid / non-essential amino acid ratio (EAA / NEAA) should be greater than 0.6. According to Table 5, the amino acid composition of *Protozoa 1* conforms to the WHO / FAO ideal protein reference model; and the lysine content of *Protozoa 1* is 2.06%, and the methionine content is 0.76%, which meets the standard requirements for the first or second limiting amino acids in most fish feeds (grass carp compound feed (GB / T 36205-2018), lysine ≥ 1.6%).

[0086] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of Achromobacterium and Protozoa in wastewater treatment, characterized by: The colorless bacillus is used in combination with protozoan algae for wastewater treatment, and the colorless bacillus is colorless bacillus ( Achromobacter xylosoxidans subsp. denitrificans AD-Z, the achromobacterium AD-Z, was deposited on September 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28350; the protochalcogenide is a protochalcogenide (… Auxenochlorella protothecoides H34#, the described protozoan was deposited on November 2, 2022 at the China General Microbiological Culture Collection Center, with accession number: CGMCC No.45371.

2. The application according to claim 1, characterized in that: Application in removing ammonia nitrogen, nitrate nitrogen, phosphorus and COD from wastewater.

3. The application according to claim 1 or 2, characterized in that: The conditions for the application are: ambient temperature controlled at 20℃~30℃, light intensity controlled at 5000lx, and light-dark cycle of 12h:12h.

4. The application of *Achromobacterium* in increasing the amino acid content of *Protozoa*; and / or the application of *Achromobacterium* in increasing the proportion of essential amino acids in *Protozoa*, characterized in that: The achromobacterium is an achromobacterium ( Achromobacter xylosoxidans subsp. denitrificans AD-Z, the achromobacterium AD-Z, was deposited on September 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28350; the protochalcogenide is a protochalcogenide (… Auxenochlorella protothecoides H34#, the described protozoan was deposited on November 2, 2022 at the China General Microbiological Culture Collection Center, with accession number: CGMCC No.45371.

5. The application according to claim 4, characterized in that: The application method includes: inoculating the colorless bacillus and the protozoan into the same environment for co-culture.

Images