Algae growth-promoting bacterium and application thereof in promoting growth of microalgae and cooperatively treating anaerobic wastewater in duck farm with microalgae

Abstract

The invention belongs to the technical field of sewage treatment, and particularly discloses an algal growth-promoting bacterium and application thereof in promoting growth of microalgae and cooperatively treating duck farm anaerobic wastewater with the microalgae, the algal growth-promoting bacterium is Citrobacter Portualensis PA1, is preserved in Guangdong Microbial Culture Collection Center on September 16, 2025, and has a preservation number of GDMCC NO: 66969, and the algal growth-promoting bacterium is preserved in the Guangdong Microbial Culture Collection Center on September 16, 2025, and is preserved in the Guangdong Microbial Culture Collection Center on September 16, 2025, and the preservation number is GDMCC NO: 66969. The algae growth-promoting bacterium is applied to promotion of microalgae growth and cooperative treatment of duck farm anaerobic wastewater with microalgae. Compared with the prior art, the combination of the algae growth-promoting bacterium and the microalgae has a better purification effect on anaerobic sewage in a duck farm than single microalgae treatment, and the bacterial strain has a good promotion effect on the growth of the microalgae and can play a better effect in practical application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology and aquaculture water environment improvement technology, and particularly relates to an algal growth-promoting bacterium and application of the algal growth-promoting bacterium in promoting growth of microalgae and treating duck farm anaerobic wastewater in cooperation with microalgae. BACKGROUND

[0002] With the increasing environmental protection requirements and intensifying competition in the livestock and poultry breeding industry, the traditional breeding wastewater treatment technology urgently needs to be innovated and upgraded in the direction of low cost and resource utilization. Microalgae can effectively treat breeding wastewater, but there are still problems such as poor environmental tolerance, long treatment period and difficulty in harvesting in actual application. In recent years, microalgae-bacteria co-culture technology has attracted much attention. This technology combines microalgae and bacteria organically, induces the formation of cell clusters or granular structures, significantly enhances the adaptability of microalgae to the complex environment of breeding wastewater, thereby improving the wastewater purification effect and constructing a cooperative treatment system. This method is expected to break through the limitations of single microalgae treatment and significantly improve the efficiency of wastewater treatment and resource recovery.

[0003] In natural aquatic environments, microalgae do not exist independently, but form a microenvironment-algal interzone with the surrounding microbial community through the exchange of metabolites and information chemicals. In this microenvironment, microalgae and microbial communities interact with each other by regulating ecological functions and diversity, and influence each other's physiological and metabolic activities. Bacteria play a key role in the algal interzone: the algal interzone provides a niche for heterotrophic bacteria, which use algal photosynthetic products while also providing beneficial functions for microalgae, promoting microalgae metabolism and biomass accumulation, forming a mutualistic symbiotic relationship. This system is defined as a microalgae-bacteria symbiotic system (MBS), in which bacteria colonized on the surface of microalgae are called microalgae particle-attached (PA) bacteria.

[0004] In the field of wastewater treatment, MBS has become a research hotspot. A large number of studies have shown that MBS performs excellently in treating actual wastewater. For example, when four different microalgae are used to treat brewery wastewater, MBS significantly outperforms single-algal treatment in COD removal rate, which only reaches 40%, while the MBS treatment group can reach more than 80%.

[0005] For example, the method for efficient treatment of livestock wastewater by synergistic interaction of bacteria and algae disclosed in Chinese patent application CN201810048400.8 uses a combination of bacteria and algae to treat livestock wastewater. Specifically, autotrophic ammonia-oxidizing bacteria, autotrophic nitrifying bacteria, and heterotrophic nitrifying bacteria work synergistically to convert ammonia nitrogen in livestock wastewater into nitrate nitrogen. Then, the treated wastewater is fed into an airlift columnar photobioreactor, where microalgae with accession number CCTCC NO:M 2017461 are used to remove nitrogen and phosphorus and accumulate biomass. Through the synergistic effect of functional microorganisms and microalgae, pollutants such as nitrogen and phosphorus in livestock wastewater are rapidly reduced, the hydraulic retention time is shortened, and the rate of microalgae biomass accumulation is increased, thereby obtaining a large amount of renewable energy while treating livestock wastewater.

[0006] For example, Chinese patent application CN201610910762.4 discloses a highly efficient Bacillus amyloliquefaciens strain for purifying aquaculture wastewater and its application. The strain, Bacillus amyloliquefaciens Am11, was deposited on February 1, 2016, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.12123. Bacillus amyloliquefaciens Am11 has strong extracellular protease activity. This strain has a significant effect on purifying water bodies and can be widely used in the aquaculture industry to reduce the use of antibiotics, thereby alleviating water pollution and increasing the benefits for farmers.

[0007] Therefore, it is of great significance to find new strains that can efficiently combine with microalgae, enhance water purification capabilities, and promote microalgae growth. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides an algae-promoting bacterium and its application in promoting microalgae growth and co-treating anaerobic wastewater from duck farms with microalgae, thereby achieving efficient removal of pollutants and simultaneous promotion of microalgae growth.

[0009] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0010] The first technical solution provided by this invention is an algae-promoting bacterium, namely Citrobacter portucalensis PA1, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 16, 2025, with the accession number GDMCC NO: 66969.

[0011] The second technical solution provided by this invention is the application of the above-mentioned algae-promoting bacteria in promoting microalgae growth and co-treating anaerobic wastewater from duck farms with microalgae.

[0012] Preferably, the microalgae is Chlorella.

[0013] Compared with existing technologies, the combination of algae-promoting bacteria and microalgae in this invention has a better purification effect on anaerobic wastewater from duck farms than microalgae treatment alone. This strain has a good promoting effect on microalgae growth and can play a good role in practical applications. Attached Figure Description

[0014] Figure 1 The changes in NH4+-N, COD, TN and TP content in UASB wastewater from a sterilized duck farm over 7 days: a) NH4+-N; b) COD; c) TN; d) TP.

[0015] Figure 2 The removal rates of NH4+-N, COD, TN and TP in the UASB wastewater from the sterilized duck farm after 7 days.

[0016] Figure 3 The following indicators were used to measure the growth of Chlorella in the UASB wastewater from the sterilized duck farm over 7 days: a) Chlorella growth density; b) Chlorella chlorophyll content on day 7; and c) Chlorella biomass.

[0017] Figure 4 For the pollutant removal rate and biomass of UASB wastewater from a real duck farm on day 7: a) pollutant removal rate; b) Chlorella biomass. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0019] Example 1: Screening and identification of Citrobacter portucalensis PA1

[0020] In this embodiment, a nitrification liquid culture medium and a nitrification agar culture medium with ammonia nitrogen as the sole nitrogen source are first prepared, and Chlorella algae solution and effluent from the primary A pond of the duck farm are added and co-cultured in the nitrification liquid culture medium for 24 h.

[0021] Subsequently, the enriched nitrified medium was filtered through a 3.0 μm polycarbonate filter membrane to retain microalgal cells, algal debris, and PA bacteria attached to them. To screen for microalgal synergistic growth-promoting bacteria with ammonia nitrogen degradation capabilities, BTB (bromothymol blue) medium was used for colorimetric reaction in this embodiment. Bacteria capable of degrading ammonia nitrogen appear blue on BTB medium. Therefore, the filter membrane was washed with PBS buffer, and the eluent was spread onto BTB agar medium using the dilution coating method. 200 PA bacteria strains that showed color were selected and inoculated into nitrified liquid medium, and then placed in a 25°C biochemical incubator for purification culture.

[0022] Subsequently, the ammonia nitrogen degradation capacity of these 200 ammonia nitrogen-degrading bacteria was determined over 48 hours: First, the ammonia nitrogen content of the initial liquid nitrification medium was measured. After dispensing the medium, the bacterial solution of each purified strain was added to a separate nitrification medium. After culturing for 48 hours, the supernatant was removed by centrifugation, and the ammonia nitrogen content was measured. Then, the degradation rate was calculated, and a PA bacterium with the best ammonia nitrogen degradation capacity was screened out, with an ammonia nitrogen degradation rate of 64.43 ± 1.20%. The ammonia nitrogen content was determined according to the method of "Methods for Monitoring and Analysis of Water and Wastewater" (Editorial Committee of Methods for Monitoring and Analysis of Water and Wastewater, State Environmental Protection Administration, 2002).

[0023] The selected PA bacterium was then identified at the molecular level using 16S rDNA sequencing. The sequencing results are as follows:

[0024]

[0025] The obtained sequencing results were compared with BLAST sequence and it was found that the strain belongs to Citrobacter portucalensis. It was named Citrobacter portucalensis PA1 and deposited at Guangdong Provincial Microbial Culture Collection Center on September 16, 2025, with accession number GDMCC NO: 66969.

[0026] Example 2: Application of Citrobacter portucalensis PA1 in promoting microalgae growth and co-treating anaerobic wastewater from duck farms

[0027] The screened Citrobacter portucalensis PA1 was combined with Chlorella to treat UASB wastewater from a sterilized duck farm. Samples were taken on days 0, 1, 2, 3, 4, 5, 6, and 7 to determine the physicochemical properties (ammonia nitrogen, COD, total nitrogen, and total phosphorus) of the wastewater, as well as the growth properties (biomass, chlorophyll content, and cell density) of the microalgae. In addition, the screened Citrobacter portucalensis PA1 was combined with Chlorella to treat UASB wastewater from a real duck farm. Samples were taken after 7 days to determine the physicochemical properties (ammonia nitrogen, COD, total nitrogen, and total phosphorus) of the wastewater and the biomass of Chlorella. The experimental groups for the sterilized duck farm UASB wastewater are shown in Table 1, and the experimental groups for the real duck farm UASB wastewater are shown in Table 2.

[0028] Table 1

[0029] Table 2

[0030] 1. After UASB wastewater from a sterilized duck farm was treated with PA1 and Chlorella in synergistic therapy, the physicochemical indicators of the wastewater (ammonia nitrogen, COD, total nitrogen, and total phosphorus), and the growth status of Chlorella (biomass content, chlorophyll content, and cell density) were measured as follows:

[0031] 1) Determination of physicochemical indicators of water quality

[0032] After passing the water sample through a 0.45 μm aqueous filter membrane, the contents of COD, total nitrogen, total phosphorus, and ammonia nitrogen were determined. The determination methods were in accordance with "Methods for Monitoring and Analysis of Water and Wastewater" (Editorial Committee of Water and Wastewater Detection and Analysis Methods, State Environmental Protection Administration, 2002); NH4 in the UASB wastewater from the duck farm was measured within 7 days. + Changes in -N, COD, TN, and TP content are as follows:Figure 1 As shown, NH4 in the UASB wastewater from the sterilized duck farm after 7 days + -N, COD, TN and TP removal rates are as follows Figure 2 As shown.

[0033] Depend on Figure 1 It can be seen that within 7 days of treatment, the concentrations of ammonia nitrogen, COD, total nitrogen and total phosphorus gradually decreased over time, and the change trend of total nitrogen was similar to that of ammonia nitrogen.

[0034] Depend on Figure 2 It was found that after 7 days, the removal rates of ammonia nitrogen, COD, total nitrogen, and total phosphorus in group A (Chlorella + PA1) of the bacterial-algae symbiotic system were 89.44% ± 1.15%, 73.65% ± 1.31%, 90.50% ± 0.58%, and 97.69 ± 0.36%, respectively, all significantly higher than those in group CK1 (single algae treatment) (P < 0.05). Therefore, it can be concluded that Citrobacter portucalensis PA1 in this embodiment can enhance the pollutant removal capacity of Chlorella.

[0035] 2) Measurement of Chlorella growth indicators

[0036] In this embodiment, the biomass of Chlorella was determined using both optical density and dry weight methods.

[0037] (1) Density method: Take 3 mL of sample and measure the absorbance at 680 nm using a UV-Vis spectrophotometer to plot the growth curve of Chlorella.

[0038] (2) Dry weight method: Take 1 mL of Chlorella sample to determine the content of chlorophyll a (Chl-a), chlorophyll b (Chl-b) and carotenoids. Centrifuge 1 mL of Chlorella sample at 4 ℃ and 8000 rpm for 15 min, remove the supernatant, rinse repeatedly with deionized water, add 1 mL of 90% acetone and store in a refrigerator at 4 ℃ protected from light for 24 h, then centrifuge at 4 ℃ and 8000 rpm for 15 min, take the supernatant and measure the absorbance at 665 nm, 652 nm and 470 nm with a UV-Vis spectrophotometer, and calculate the content of chlorophyll a, chlorophyll b and carotenoids according to the following formula:

[0039] Chlorophyll a (mg / L) = 16.72A 665 -9.16A 652 ;

[0040] Chlorophyll b (mg / L) = 34.09A 652 -15.28A 665 ;

[0041] Carotenoids (mg / L) = (1000 × A) 470 - 1.63 × Chl - a) / 221;

[0042] Biomass was determined using the dry weight method. A 5 mL sample of *Chlorella vulgaris* was taken and filtered through a dried 0.45 μm aqueous membrane (m0). After filtration, the membrane and sample were placed in a 60 ℃ oven and dried to constant weight, then weighed (m1). Where: ; In the formula: the dry weight unit is g / L;

[0043] m0 is the weight of the dried filter membrane, in grams;

[0044] m1 is the weight of the dried filter membrane and algae, in grams;

[0045] V represents the dry weight sample volume, in L;

[0046] This embodiment measured the growth density of Chlorella within 7 days, the chlorophyll content of Chlorella on day 7, and the biomass of Chlorella. The results are as follows: Figure 3 As shown. By Figure 3 The results showed that group A (Chlorella + PA1 bacteria) exhibited the best growth in the algal symbiotic system experiment. In group CK1, Chlorella growth was initially inhibited by wastewater, but improved starting from day 5. Except for chlorophyll b, which showed no significant difference, all indicators in group A were significantly higher than those in group CK1 (single algae treatment) (P < 0.05). Specifically, the total chlorophyll, carotenoids, and biomass in group A reached 2.51 ± 0.36 mg / L, 0.46 ± 0.09 mg / L, and 13.8 ± 1.7 g / L, respectively, all significantly higher than those in group CK1 (P < 0.05). These results indicate that PA1 bacteria have a significant promoting effect on Chlorella growth.

[0047] 2. The physicochemical indicators (ammonia nitrogen, COD, total nitrogen, and total phosphorus) of UASB wastewater from a real duck farm after co-treatment with PA1 and Chlorella were as follows: The growth of Chlorella (biomass content, chlorophyll content, and cell density) was also measured.

[0048] The pollutant removal rate and biomass of UASB wastewater from a real duck farm on day 7 are as follows: Figure 4 As shown. By Figure 4The results showed that the removal rates of ammonia nitrogen, COD, total nitrogen, and total phosphorus, and the total biomass of group B (Chlorella + PA1 bacteria) in the algae-bacteria symbiotic system were 68.35% ± 1.55%, 77.4% ± 2.58%, 77.35% ± 3.37%, 55.41% ± 8.81%, and 4.73 ± 0.55 g / L, respectively, all significantly higher than those of group CK2 (single algae treatment) (P < 0.05). These results indicate that the algae-bacteria symbiotic system is still superior to Chlorella alone in purifying real wastewater, but compared to the UASB wastewater from a duck farm, the treatment efficiency of the algae-bacteria symbiotic system for ammonia nitrogen, total nitrogen, and total phosphorus was slightly lower (comparison). Figure 3 However, the removal rates of COD, ammonia nitrogen, and total nitrogen were still >68%; at the same time, the biomass of Chlorella was also significantly higher than that of the single algae treatment group (CK2).

[0049] In summary, the combination of algae-promoting bacteria and microalgae in this invention has a better purification effect on anaerobic wastewater from duck farms than microalgae treatment alone. This strain has a good promoting effect on microalgae growth and can play a good role in practical applications.

[0050] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An algae-promoting bacterium, characterized in that, The algae-promoting bacteria is Citrobacter lucis ( ) Citrobacter portucalensis PA1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 16, 2025, with accession number GDMCC NO: 66969.

2. The application of the algae-promoting bacteria as described in claim 1 in promoting microalgae growth and synergistically treating anaerobic wastewater from duck farms with microalgae.

3. The application according to claim 2, characterized in that: The microalgae mentioned is Chlorella vulgaris.

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