Construction method of microalgae-in-situ bacteria strengthening system for degrading naphthenic acid

Abstract

The invention is applicable to the technical field of industrial wastewater treatment, and provides a construction method of a microalgae-in-situ bacteria strengthening system for degrading naphthenic acid, which comprises the following steps: separating and identifying two strains of in-situ bacteria from a discharge port of an oil-producing wastewater treatment plant, and constructing a three-strain typical stain-resistant green alga-in-situ bacteria co-culture system. The strengthening system can highly tolerate and efficiently degrade naphthenic acid, can effectively remove nitrogen and phosphorus, and has higher biomass and high-value compound yield. The microalgae-in-situ bacterium strengthening system can reduce pollutant discharge and improve the microalgae resource utilization rate, and the microalgae-in-situ bacterium strengthening system can be used as a biological strengthening degradation technology of naphthenic acid and can be suitable for purification treatment of oil production wastewater and petroleum polluted water areas.

Description

Technical Field

[0001] This invention belongs to the field of industrial wastewater treatment technology, and in particular relates to a method for constructing a microalgae-in-situ bacteria enhancement system for degrading naphthenic acids. Background Technology

[0002] Naphthenic acids, a natural component of petroleum, have become a significant pollutant in various petroleum industry wastewaters. Utilizing algal-microbial symbiotic systems to remediate naphthenic acid wastewater can reduce the biotoxicity of naphthenic acids, effectively remove carbon, nitrogen, and phosphorus from the wastewater, and increase its own biomass through photosynthetic carbon fixation, thus contributing to carbon emission reduction. Furthermore, its biomass can become a green raw material for biofuels or functional oils, demonstrating high potential for resource utilization.

[0003] To improve the feasibility of using algal-bacterial symbiosis to efficiently degrade naphthenic acid, the selected algal strains and bacterial strains must meet the following three conditions: (1) The selected algal strains and bacterial strains can be used in situ in naphthenic acid wastewater (approximately 100 mg / L) -1 (1) The algae can tolerate the toxicity of naphthenic acid in wastewater with a concentration of naphthenic acid or higher; (2) The algae have a relatively fast growth rate to maintain a certain production volume; (3) The algae or algal strains have the ability to reduce the concentration of naphthenic acid in in-situ through absorption or degradation processes.

[0004] Under low-light, nitrogen-rich standard growth conditions, exposure to cyclohexanecarboxylic acid (CRA) significantly inhibited the growth and photosynthetic activity of the model green alga *Chlamydomonas reinhardtii* in a low initial density culture system, and also significantly altered its fatty acid composition. Furthermore, in a high initial density culture system, high-light, nitrogen-rich conditions enhanced *Chlamydomonas reinhardtii*'s tolerance to CRA by promoting phosphorus absorption and altering fatty acid composition; while high-light, nitrogen-limited conditions weakened its tolerance to CRA by inhibiting photosynthetic activity and phosphorus absorption. However, there are currently no reports on the construction of enhanced systems for in-situ co-culture of *Chlamydomonas reinhardtii* or other pollution-tolerant microalgae with CRA-derived bacteria from CRA-derived wastewater, nor on their tolerance and degradation capabilities to CRA. Therefore, there is an urgent need to develop microalgae-in-situ bacteria enhanced systems that can highly tolerate and efficiently degrade CRA, thereby providing high-quality algal / bacterial resources for the treatment of large-scale CRA-derived wastewater using bio-enhanced degradation methods. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acids, thereby addressing the problems mentioned in the background art.

[0006] The present invention is implemented as follows: a method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acids includes the following steps:

[0007] Step 1: Two in-situ bacteria strains from the discharge outlet of the oilfield wastewater treatment plant were isolated and identified. These strains included *Pseudomonas mendocina* (strain B2) and *Chryseoglobus* sp. (strain B4). Three fast-growing, pollution-tolerant green algae strains—*Chlamydomonas reinhardtii*, *Chlorella pyrenoidosa*, and *Tetradesmus obliquus*—were selected and co-cultured in these systems at a microalgae:bacteria ratio of 10:1 (optical density value). The light intensity was 100 μmol / (m²). 2 / s), the photoperiod is light:dark = 12h:12h, shake or stand still for 6 days, and multi-trophic culture is carried out in TAP medium with NaOH or cyclohexanecarboxylic acid (CHCA) added, the culture volume is 200mL, and the culture temperature is 25±3℃.

[0008] Step 2: Monitor the growth indicators, photosynthetic activity, nitrogen and phosphorus concentration changes, biochemical components and CHCA degradation rate of the pollution-tolerant green algae-in-situ bacteria co-culture system. Comprehensively evaluate the tolerance, degradation, nitrogen and phosphorus removal performance and resource utilization rate of the pollution-tolerant green algae-in-situ bacteria co-culture system to naphthenic acid, and finally screen out the optimal microalgae-in-situ bacteria enhancement system.

[0009] In a further technical solution, in step 1, the in-situ bacteria are isolated and purified from the discharge outlet of the oilfield wastewater treatment plant. The two selected in-situ bacteria are obtained based on their growth rate, their impact on microalgae growth, and their degradation rate of CHCA.

[0010] In a further technical solution, in step 1, the Chlamydomonas reinhardtii strain is an oil-accumulating starch synthesis defective strain, purchased from the Chlamydomonas reinhardtii Center in the United States. Due to the lack of cell walls and flagella, it needs to be cultured on a shaker at a speed of 130 rpm.

[0011] Chlorella proteoglycans were purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences, while Scenedesmus obliquus was obtained by separation and purification from citric acid wastewater.

[0012] Chlorella proteoglycans and Scenedesmus obliqueis are more suitable for static culture, with the flask shaken 3 times a day.

[0013] In a further technical solution, in step 1, before 6 days of shaking or static culture, three pollution-tolerant microalgae strains are pre-cultured for 3 days under a light intensity of 100 μmol / (m²). 2 / s), with a photoperiod of light:dark = 12h:12h, to obtain a sufficient number of algal cells in the exponential growth phase, with an initial inoculation density of approximately 1×10 5 per mL.

[0014] In a further technical solution, in step 1, the co-solvent for CHCA is 0.1 mol / L NaOH, the control group is algal solution or algae-bacteria co-culture solution in TAP medium with 0.1 mol / L NaOH, and the experimental group is algal solution or algae-bacteria co-culture solution in TAP medium with 100 mg / L CHCA.

[0015] In a further technical solution, in step 2, the growth indicators include the optical density value OD. 750 Dry weight density and pigment concentration were determined by the mass difference method and the ethanol extraction method, respectively.

[0016] Photosynthetic activity includes the maximum photon conversion efficiency, actual photon conversion efficiency, and electron transfer rate of photosystem II; nitrogen and phosphorus concentrations were determined by Nessler's reagent method and ammonium molybdate spectrophotometry, respectively.

[0017] In a further technical solution, in step 2, the biochemical components include carbohydrates, proteins, glycerides and their acyl groups. The yield of the above-mentioned high-value compounds in each experimental group is further calculated based on the dry weight density within 6 days of cultivation, expressed in mg / (L / d), to evaluate the resource utilization efficiency of the algae-bacteria co-culture system.

[0018] In a further technical solution, in step 2, the CHCA degradation rate is obtained by chloroform extraction combined with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization, and CHCA is quantitatively analyzed by gas chromatography based on the internal standard method.

[0019] This invention provides a method for constructing a microalgae-in-situ bacteria-enhanced system for degrading naphthenic acids. The constructed algae-bacteria co-culture system can highly tolerate and efficiently degrade typical naphthenic acids (CHCA), with a CHCA degradation rate as high as 68% and nitrogen and phosphorus absorption efficiencies as high as 75% and 57%, respectively. It can effectively remove nitrogen and phosphorus elements from oilfield wastewater. While removing pollutants, it can fix inorganic and organic carbon in the water into biomass. The synthesized high-value compounds such as carbohydrates, proteins, and glycerides can be further utilized for resource recovery, contributing to carbon emission reduction. Attached Figure Description

[0020] Figure 1 Phylogenetic analysis diagram of 16S rRNA gene sequences of four in situ bacteria;

[0021] Figure 2 The effect of CHCA exposure on the color of the culture medium in the co-culture system of Chlamydomonas reinhardtii strain 4;

[0022] Figure 3The effects of CHCA exposure on the growth and photosynthetic activity of Chlamydomonas reinhardtii-4 in situ bacterial co-culture system;

[0023] Figure 4 The effect of CHCA exposure on nitrogen and phosphorus uptake in the co-culture system of Chlamydomonas reinhardtii-4 strains of bacteria;

[0024] Figure 5 The effect of CHCA exposure on the biochemical component content of the in situ co-culture system of Chlamydomonas reinhardtii-4 strain;

[0025] Figure 6 The effect of CHCA exposure on fatty acid composition in the co-culture system of Chlamydomonas reinhardtii strain 4;

[0026] Figure 7 Phylogenetic analysis diagram of the 18S rRNA gene sequence of Scenedesmus obliquus;

[0027] Figure 8 The effects of CHCA exposure on growth, photosynthetic activity, and nitrogen and phosphorus uptake in a co-culture system of Chlorella proteoglycans and Scenedesmus obliquus-2 strains of in situ bacteria;

[0028] Figure 9 The effect of CHCA exposure on the content of biochemical components in a co-culture system of Chlorella proteoidis and Scenedesmus obliquus-2 strains of in situ bacteria;

[0029] Figure 10 The effect of CHCA exposure on fatty acid composition in a co-culture system of Chlorella proteoglycans and Scenedesmus obliquus-2 strains of in situ bacteria;

[0030] Figure 11 The effect of CHCA exposure on the yield of biomass and high-value compounds in a co-culture system of Chlorella proteoidis and Scenedesmus obliquus-2 strains of in situ bacteria;

[0031] Figure 12 Comparison of CHCA degradation rates in co-culture systems of Chlamydomonas reinhardtii, Chlorella proteoglycans, and Scenedesmus obliquus-2 strains of in situ bacteria. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0034] An embodiment of the present invention provides a method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acids. The method includes the following steps:

[0035] Step 1: Two in-situ bacteria strains from the discharge outlet of the oilfield wastewater treatment plant were isolated and identified. These bacteria included *Pseudomonas mendoza* and *Coccidia cerasifera*. Three fast-growing, pollution-tolerant green algae strains, including *Chlamydomonas reinhardtii*, *Chlorella proteoglycans*, and *Scenedesmus obliquus*, were selected. Pollution-tolerant green algae-in-situ bacteria co-culture systems were constructed at a microalgae:bacteria ratio of 10:1 (optical density value) under a light intensity of 100 μmol / (m²). 2 / s), the photoperiod is light:dark = 12h:12h, shake or stand still for 6 days, and multi-trophic culture is carried out in TAP medium with NaOH or CHCA, the culture volume is 200mL, and the culture temperature is 25±3℃.

[0036] Step 2: Monitor the growth indicators, photosynthetic activity, nitrogen and phosphorus concentration changes, biochemical components and CHCA degradation rate of the pollution-tolerant green algae-in-situ bacteria co-culture system. Comprehensively evaluate the tolerance, degradation, nitrogen and phosphorus removal performance and resource utilization rate of the pollution-tolerant green algae-in-situ bacteria co-culture system to naphthenic acid, and finally screen out the optimal microalgae-in-situ bacteria enhancement system.

[0037] In a preferred embodiment of the present invention, in step 1, the in-situ bacteria are isolated and purified from the discharge outlet of the oilfield wastewater treatment plant. The two selected in-situ bacteria are obtained based on their growth rate, their effect on microalgae growth, and their degradation rate of CHCA.

[0038] In a preferred embodiment of the present invention, in step 1, the Chlamydomonas reinhardtii strain is an oil-accumulating starch synthesis defective strain, purchased from the Chlamydomonas reinhardtii Center in the United States. Due to the lack of cell walls and flagella, it needs to be cultured on a shaker at a speed of 130 rpm.

[0039] Chlorella proteoglycans were purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences, while Scenedesmus obliquus was obtained by separation and purification from citric acid wastewater.

[0040] Chlorella proteoglycans and Scenedesmus obliqueis are more suitable for static culture, with the flask shaken 3 times a day.

[0041] In a preferred embodiment of the present invention, in step 1, before 6 days of shaking or static culture, three strains of pollution-tolerant microalgae are pre-cultured for 3 days under a light intensity of 100 μmol / (m²). 2 / s), with a photoperiod of light:dark = 12h:12h, to obtain a sufficient number of algal cells in the exponential growth phase, with an initial inoculation density of approximately 1×10 5 per mL.

[0042] In a preferred embodiment of the present invention, in step 1, the co-solvent for CHCA is 0.1 mol / L NaOH, the control group is algal solution or algae-bacteria co-culture solution in TAP medium with 0.1 mol / L NaOH, and the experimental group is algal solution or algae-bacteria co-culture solution in TAP medium with 100 mg / L CHCA.

[0043] In a preferred embodiment of the present invention, in step 2, the growth index includes the optical density value OD. 750 Dry weight density and pigment concentration were determined by the mass difference method and the ethanol extraction method, respectively.

[0044] Photosynthetic activity includes the maximum photon conversion efficiency, actual photon conversion efficiency, and electron transfer rate of photosystem II; nitrogen and phosphorus concentrations were determined by Nessler's reagent method and ammonium molybdate spectrophotometry, respectively.

[0045] In a preferred embodiment of the present invention, in step 2, the biochemical components include carbohydrates, proteins, glycerides and their acyl groups. The yield of the above-mentioned high-value compounds in each experimental group is further calculated based on the dry weight density within 6 days of cultivation, expressed in mg / (L / d), to evaluate the resource utilization efficiency of the algae-bacteria co-culture system.

[0046] In a preferred embodiment of the present invention, in step 2, the CHCA degradation rate is obtained by chloroform extraction combined with derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and the CHCA is quantitatively analyzed by gas chromatography based on the internal standard method.

[0047] Several specific embodiments are provided below to verify the effectiveness of this method.

[0048] Example 1: Construction of a co-culture system of Chlamydomonas reinhardtii and 4 in situ bacteria;

[0049] The effects of CHCA exposure on growth parameters, photosynthetic activity, nitrogen and phosphorus uptake, biochemical component content, and fatty acid composition of a co-culture system of *Chlamydomonas reinhardtii* and four in situ bacterial strains were evaluated. *Chlamydomonas reinhardtii* in its exponential growth phase and bacteria were mixed at an algae-to-bacteria ratio of 10:1 based on optical density. The culture was conducted for 6 days at 25.0 ± 2.0 °C, a 12-hour light:12-hour dark photoperiod, a light intensity of 2000 lux, and continuous shaking (120 rpm). The bacteria used to construct the algae-bacteria co-culture system included *Pseudomonas mendocina* (B2), *Pseudomonas toyotomiensis* (B3), *Chryseoglobus* sp. (B4), and *Candidatus Rhizobium massiliae* (B5). See details below. Figure 1 (Where A represents B2; B represents B3; C represents B4; and D represents rhizobia). 10-15 mL of culture medium from each treatment group was collected daily for OD measurement. 750 Photosynthetic activity and nitrogen and phosphorus concentrations. On day 6 (see details...) Figure 2 All microbial cells were collected by centrifugation (6000 rpm, 4°C), with three biological replicates. The collected microbial cell samples were rapidly frozen after centrifugation for subsequent determination of biochemical components, including lipid, carbohydrate, and protein content, as well as fatty acid composition.

[0050] Under CHCA exposure conditions, strain B4 had the least impact on the growth of *Chlamydomonas reinhardtii*, followed by B2 and B5. B3 showed the most significant inhibitory effect on *Chlamydomonas reinhardtii* growth. (See details...) Figure 2 and Figure 3 (Where, A represents the OD of the Chlamydomonas reinhardtii + B2 co-culture system) 750 B represents the photosynthetic activity of the *Chlamydomonas reinhardtii* + B2 co-culture system, and C represents the OD of the *Chlamydomonas reinhardtii* + B3 co-culture system. 750 D represents the photosynthetic activity of the *Chlamydomonas reinhardtii* + B3 co-culture system, and E represents the OD of the *Chlamydomonas reinhardtii* + B4 co-culture system. 750 F represents the photosynthetic activity of the *Chlamydomonas reinhardtii* + B4 co-culture system, and G represents the OD of the *Chlamydomonas reinhardtii* + B5 co-culture system. 750 H represents the photosynthetic activity of the *Chlamydomonas reinhardtii* + B5 co-culture system; strain B2 significantly promoted the photosynthetic activity of *Chlamydomonas reinhardtii*, while the other three in situ bacteria significantly inhibited the photosynthetic activity of *Chlamydomonas reinhardtii* (see details). Figure 3 ).

[0051] Under CHCA exposure conditions, strains B2-B5 did not significantly affect nitrogen and phosphorus uptake by *Chlamydomonas reinhardtii* (see details). Figure 4Where A represents the nitrogen uptake rate of the *Chlamydomonas reinhardtii* + B2 co-culture system, B represents the phosphorus uptake rate of the *Chlamydomonas reinhardtii* + B2 co-culture system, C represents the nitrogen uptake rate of the *Chlamydomonas reinhardtii* + B3 co-culture system, D represents the phosphorus uptake rate of the *Chlamydomonas reinhardtii* + B3 co-culture system, E represents the nitrogen uptake rate of the *Chlamydomonas reinhardtii* + B4 co-culture system, F represents the phosphorus uptake rate of the *Chlamydomonas reinhardtii* + B4 co-culture system, G represents the nitrogen uptake rate of the *Chlamydomonas reinhardtii* + B5 co-culture system, and H represents the phosphorus uptake rate of the *Chlamydomonas reinhardtii* + B5 co-culture system.

[0052] Under CHCA exposure conditions, strain B2 had no significant effect on the glycerol, carbohydrate, and protein content of *Chlamydomonas reinhardtii*. Strains B3 and B5 significantly reduced the glycerol content of *Chlamydomonas reinhardtii*, strain B4 significantly promoted carbohydrate accumulation, and strain B5 significantly promoted protein accumulation (see details). Figure 5 Where A represents the glycerol content of the *Chlamydomonas reinhardtii* + B2 co-culture system, B represents the carbohydrate content of the *Chlamydomonas reinhardtii* + B2 co-culture system, C represents the protein content of the *Chlamydomonas reinhardtii* + B2 co-culture system, D represents the glycerol content of the *Chlamydomonas reinhardtii* + B3 co-culture system, E represents the carbohydrate content of the *Chlamydomonas reinhardtii* + B3 co-culture system, F represents the protein content of the *Chlamydomonas reinhardtii* + B3 co-culture system, G represents the glycerol content of the *Chlamydomonas reinhardtii* + B4 co-culture system, H represents the carbohydrate content of the *Chlamydomonas reinhardtii* + B4 co-culture system, I represents the protein content of the *Chlamydomonas reinhardtii* + B4 co-culture system, J represents the glycerol content of the *Chlamydomonas reinhardtii* + B5 co-culture system, K represents the carbohydrate content of the *Chlamydomonas reinhardtii* + B5 co-culture system, and L represents the protein content of the *Chlamydomonas reinhardtii* + B5 co-culture system.

[0053] Under CHCA exposure conditions, strains B2 and B4 did not significantly alter the fatty acid composition of *Chlamydomonas reinhardtii*. Strains B3 and B5 significantly reduced the relative abundance of saturated fatty acid C16:0 and polyunsaturated fatty acids C16:4n3, C18:2n6, C18:3n6, and C18:3n3, while significantly increasing the relative abundance of monounsaturated fatty acid C18:1n7 (see details). Figure 6 Where A represents the fatty acid composition of the *Chlamydomonas reinhardtii* + B2 co-culture system, B represents the fatty acid composition of the *Chlamydomonas reinhardtii* + B3 co-culture system, C represents the fatty acid composition of the *Chlamydomonas reinhardtii* + B4 co-culture system, and D represents the fatty acid composition of the *Chlamydomonas reinhardtii* + B5 co-culture system.

[0054] Based on the above results, two co-culture systems, Chlamydomonas reinhardtii-B2 and Chlamydomonas reinhardtii-B4, were selected. These two algae-bacteria co-culture systems have higher tolerance to CHCA and higher potential for resource utilization.

[0055] Example 2: Construction of a co-culture system of pollution-tolerant green algae and two in situ bacteria: Chlorella pyrenoidosa or Scenedesmus obliquus in the exponential growth phase (see details) Figure 7 Algae and bacteria were mixed at an algae-to-bacteria ratio of 10:1. The culture was conducted at 25.0±2.0℃ with a 12h light:12h dark photoperiod and a light intensity of 2000 lux for 6 days under static incubation conditions. 10-15 mL of culture medium from each treatment group was collected daily or every two days for parameter determination, including growth indicators, photosynthetic activity, and nitrogen and phosphorus concentrations. On day 6, all microbial cells were collected by centrifugation (6000 rpm, 4℃), with three biological replicates. The collected microbial cell samples were centrifuged and then rapidly frozen for subsequent determination of biochemical components, including lipid, carbohydrate, and protein content, as well as fatty acid composition.

[0056] Under CHCA exposure conditions, strain B4 significantly inhibited the growth of *Chlorella protozoa*, while strain B2 had no significant effect on its growth; strains B2 and B4 did not significantly affect the growth of *Scenedesmus obliquus* (see details). Figure 8 Where A is the OD of the Chlorella proteoglycans + B2 / B4 co-culture system. 750 B represents the OD of the *Scenedesmus obliquus* co-culture system with B2 / B4. 750 C represents the photosynthetic activity of the *Chlorella proteoglycans* + B2 / B4 co-culture system; D represents the photosynthetic activity of the *Scenedesmus obliquus* + B2 / B4 co-culture system; E represents the nitrogen uptake rate of the *Chlorella proteoglycans* + B2 / B4 co-culture system; F represents the nitrogen uptake rate of the *Scenedesmus obliquus* + B2 / B4 co-culture system; G represents the phosphorus uptake rate of the *Chlorella proteoglycans* + B2 / B4 co-culture system; and H represents the phosphorus uptake rate of the *Scenedesmus obliquus* + B2 / B4 co-culture system.

[0057] Under CHCA exposure conditions, strains B2 and B4 did not significantly affect the photosynthetic activity and nitrogen and phosphorus uptake of Chlorella vulgaris and Scenedesmus obliquus (see details). Figure 8 ).

[0058] Under CHCA exposure conditions, strain B2 had no significant effect on the glycerol, carbohydrate, and protein content of *Chlorella pyrenoidosa*; strain B4 had no significant effect on the glycerol content of *Chlorella pyrenoidosa*, but significantly promoted the decomposition of carbohydrates and proteins. Strain B2 significantly promoted the decomposition of glycerol in *Scenedesmus obliquus*, but had no significant effect on its carbohydrate and protein content; strain B4 had no significant effect on the content of the three organic carbon biochemical components of *Scenedesmus obliquus* (see details). Figure 9Where A represents the glycerol content of the Chlorella proteoglycans + B2 / B4 co-culture system, B represents the carbohydrate content of the Chlorella proteoglycans + B2 / B4 co-culture system, C represents the protein content of the Chlorella proteoglycans + B2 / B4 co-culture system, D represents the glycerol content of the Scenedesmus obliquus + B2 / B4 co-culture system, E represents the carbohydrate content of the Scenedesmus obliquus + B2 / B4 co-culture system, and F represents the protein content of the Scenedesmus obliquus + B2 / B4 co-culture system.

[0059] Under CHCA exposure conditions, strains B2 and B4 had no significant effect on the fatty acid composition of *Chlorella pyrenoidosa*. Strain B2 significantly increased the relative abundance of saturated fatty acids C16:0, monounsaturated fatty acids C16:1n7, and polyunsaturated fatty acids C16:3n3 and C18:2n6 in *Scenedesmus obliquus*, while significantly decreasing the relative abundance of monounsaturated fatty acids C18:1n9 and polyunsaturated fatty acids C16:4n3. Strain B4 had no significant effect on the fatty acid composition of *Scenedesmus obliquus* (see details). Figure 10 (Where A represents the fatty acid composition of the Chlorella proteoglycans + B2 / B4 co-culture system, and B represents the fatty acid composition of the Scenedesmus obliquus + B2 / B4 co-culture system).

[0060] Based on the above results, the co-culture systems of Chlorella proteoglycans-B2 and Scenedesmus obliquus-B4 showed higher tolerance to CHCA and greater potential for resource utilization.

[0061] Example 3: Evaluation of biomass yield, yield of three organic carbon biochemical components, and CHCA degradation rate in a co-culture system of *Chlamydomonas reinhardtii*, *Chlorella proteoglycans*, and *Scenedesmus obliquus*, as well as strains B2 and B4. Biomass yield and the yield of the three organic carbon biochemical components were calculated using the dry weight density of the algal culture medium and the contents of glycerol esters, carbohydrates, and proteins. CHCA concentration was quantitatively analyzed using chloroform extraction / BSTFA derivatization / GC determination.

[0062] Under CHCA exposure conditions, the biomass yield of the co-culture system of Chlorella proteoglycans and Scenedesmus obliquus, along with strains B2 and B4, was 3-7 times higher than that of the co-culture system of Chlamydomonas reinhardtii and strains B2 and B4 (see details). Figure 11 ).

[0063] Under CHCA exposure conditions, the glycerol yield of the pure culture group of *Scenedesmus obliquus* was twice that of *Chlamydomonas reinhardtii* and *Chlorella proteoglycans*, and the glycerol yield of the co-culture system of *Chlorella proteoglycans* and *Scenedesmus obliquus* with strain B4 was 1.4 times that of the co-culture system of *Chlamydomonas reinhardtii* and B4 (see details). Figure 11Where A represents the biomass yield of the three green algae, B represents the biomass yield of the co-culture system of the three green algae + B2, C represents the biomass yield of the co-culture system of the three green algae + B4, D represents the glycerol ester yield of the three green algae, E represents the glycerol ester yield of the co-culture system of the three green algae + B2, F represents the glycerol ester yield of the co-culture system of the three green algae + B4, G represents the carbohydrate yield of the three green algae, H represents the carbohydrate yield of the co-culture system of the three green algae + B2, I represents the carbohydrate yield of the co-culture system of the three green algae + B4, J represents the protein yield of the three green algae, K represents the protein yield of the co-culture system of the three green algae + B2, and L represents the protein yield of the co-culture system of the three green algae + B4.

[0064] Under CHCA exposure conditions, the carbohydrate yield of the pure culture group of *Scenedesmus obliquus* was 2-3 times that of *Chlamydomonas reinhardtii* and *Chlorella proteoglycans*, and the carbohydrate yield of the co-culture system of *Chlorella proteoglycans*, *Scenedesmus obliquus*, and strains B2 and B4 was 1.4-3.3 times that of the co-culture system of *Chlamydomonas reinhardtii* and strains B2 and B4 (see details). Figure 11 ).

[0065] Under CHCA exposure conditions, the protein yield of the Chlamydomonas reinhardtii-B4 co-culture system was 1.6-1.9 times that of the Chlorella vulgaris and Scenedesmus obliquus-B4 co-culture systems (see details). Figure 11 ).

[0066] The CHCA degradation results showed that strains B2 and B4 significantly enhanced the degradation of CHCA by *Chlamydomonas reinhardtii*, strain B4 significantly enhanced the degradation of CHCA by *Chlorella protozoa*, and strains B2 and B4 significantly enhanced the degradation of CHCA by *Scenedesmus obliquus*. Furthermore, the *Scenedesmus obliquus*-strains B2 and B4 co-culture system achieved a CHCA degradation rate exceeding 60%, significantly higher than other algae-bacteria co-culture systems; the *Chlamydomonas reinhardtii*-B2 and B4 and *Chlorella protozoa*-B4 co-culture systems achieved CHCA degradation rates as high as 18-22%, with the latter showing the second highest CHCA degradation performance (see details). Figure 12 Where A represents the CHCA degradation rate of the Chlamydomonas reinhardtii + B2 / B4 co-culture system, B represents the CHCA degradation rate of the Chlorella proteoglycans + B2 / B4 co-culture system, and C represents the CHCA degradation rate of the Scenedesmus obliquus + B2 / B4 co-culture system.

[0067] Based on the above results, under CHCA exposure conditions, the co-culture systems of *Chlorella pyrenoidosa* and *Scenedesmus obliquus* with strain B4 exhibited higher biomass, glycerol, and carbohydrate yields. In contrast, although the biomass yield of the *Chlamydomonas reinhardtii*-B4 co-culture system was lower than that of the *Chlorella pyrenoidosa* and *Scenedesmus obliquus* co-culture systems with strain B4, this system had a higher protein yield. Furthermore, the co-culture systems of *Scenedesmus obliquus* with strains B2 and B4 showed the highest CHCA degradation performance. In conclusion, the *Scenedesmus obliquus*-strain B4 co-culture system demonstrated higher resource utilization and the best CHCA degradation performance.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acids, characterized in that, Includes the following steps: Step 1: Two in-situ bacteria strains from the discharge outlet of the oilfield wastewater treatment plant were isolated and identified. These bacteria included *Pseudomonas mendoza* and *Coccidia cerasifera*. Three fast-growing, pollution-tolerant green algae strains, including *Chlamydomonas reinhardtii*, *Chlorella proteoglycans*, and *Scenedesmus obliquus*, were selected. Pollution-tolerant green algae-in-situ bacteria co-culture systems were constructed with a microalgae:bacteria ratio of 10:1 and an illuminance of 100 μmol / (m²). 2 / s), the photoperiod is light:dark = 12h:12h, shake or stand still for 6 days, and multi-trophic culture is carried out in TAP medium with NaOH or CHCA, the culture volume is 200mL, and the culture temperature is 25±3℃. Step 2: Monitor the growth indicators, photosynthetic activity, nitrogen and phosphorus concentration changes, biochemical components and CHCA degradation rate of the pollution-tolerant green algae-in-situ bacteria co-culture system. Comprehensively evaluate the tolerance, degradation, nitrogen and phosphorus removal performance and resource utilization rate of the pollution-tolerant green algae-in-situ bacteria co-culture system to naphthenic acid, and finally screen out the optimal microalgae-in-situ bacteria enhancement system.

2. The method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acid according to claim 1, characterized in that, In step 1, the in-situ bacteria are isolated and purified from the discharge outlet of the oilfield wastewater treatment plant. The two selected in-situ bacteria were obtained based on their growth rate, their impact on microalgae growth, and their degradation rate of CHCA.

3. The method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acids according to claim 1, characterized in that, In step 1, Chlamydomonas reinhardtii is an oil-accumulating starch synthesis defect strain and needs to be cultured on a shaker at a speed of 130 rpm. Chlorella proteoglycans were purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences, while Scenedesmus obliquus was obtained by separation and purification from citric acid wastewater. Chlorella proteoglycans and Scenedesmus obliqueis were cultured statically with the flasks shaken three times a day.

4. The method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acid according to claim 1, characterized in that, In step 1, before 6 days of shaking or static culture, three pollution-tolerant microalgae strains are pre-cultured for 3 days under a light intensity of 100 μmol / (m²). 2 / s), with a photoperiod of light:dark = 12h:12h, to obtain a sufficient number of algal cells in the exponential growth phase, with an initial inoculation density of approximately 1×10 5 per mL.

5. The method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acid according to claim 1, characterized in that, In step 2, the growth indicators include the optical density value OD. 750 Dry weight density and pigment concentration were determined by the mass difference method and the ethanol extraction method, respectively. Photosynthetic activity includes the maximum quantum conversion efficiency of photosystem II, the actual quantum conversion efficiency, and the electron transfer rate; nitrogen and phosphorus concentrations were determined using Nessler's reagent method and ammonium molybdate spectrophotometry, respectively.

6. The method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acid according to claim 1, characterized in that, In step 2, the biochemical components include carbohydrates, proteins, glycerides and their acyl groups. Based on the dry weight density, the yield of the above high-value compounds in each experimental group within 6 days of cultivation is further calculated, expressed in mg / (L / d), to evaluate the resource utilization efficiency of the algae-bacteria co-culture system.

7. The method for constructing a microalgae-in-situ bacteria enhancement system for degrading cycloalkanoic acid according to claim 1, characterized in that, In step 2, the CHCA degradation rate was determined by chloroform extraction combined with N,O-bis(trimethylsilyl)trifluoroacetamide derivatization, and CHCA was quantitatively analyzed by gas chromatography based on the internal standard method.

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