A configurable spatialized bacteria-algae integrated biophotonic system and its preparation method and application

Abstract

The present invention provides a configurable spatial bacteria-algae integrated biophoton system and a preparation method and application thereof, belonging to the technical field of wastewater treatment. The configurable spatial bacteria-algae integrated biophoton system is a transparent gel sphere with a core-shell structure, and the sphere diameter of the gel sphere is 110-120 μm; the core part of the core-shell structure contains Chlorella and a first hydrophilic carrier, and the outer shell part contains Bacillus licheniformis and a second hydrophilic carrier; the ratio of the sphere diameter of the core layer to the width of the shell layer is (0.3-3.75):1. The configurable spatial bacteria-algae integrated biophoton system is prepared by a microfluidic droplet generator. The configurable spatial bacteria-algae integrated biophoton system is used to treat wastewater, and the degradation efficiency of total nitrogen, total phosphorus and chemical oxygen demand within 72 hours reaches 89.89%, 91.8% and 91.98%, respectively.

Description

Technical Field

[0001] The present invention belongs to the technical field of wastewater treatment, and in particular relates to a configurable spatial bacteria-algae integrated biophotonic system and a preparation method and application thereof. Background Art

[0002] Global temperatures are currently rising due to the massive emission of greenhouse gases. Statistics show that energy consumption in sewage treatment plants accounts for 3% of total electricity consumption, and carbon emissions account for 1.6% of total greenhouse gas emissions. Carbon neutrality in the sewage treatment industry is an urgent issue to be addressed. The current main industrial method is to use activated sludge to remove nitrogen and phosphorus from wastewater. It usually includes a series of steps to degrade nitrogen, phosphorus and organic matter, such as aerobic, anoxic and anaerobic treatment methods. How to reduce greenhouse gas emissions, control resource waste and reduce treatment costs are urgent needs of the sewage treatment industry.

[0003] Active and direct carbon capture and utilization (CCU) and continuous pollutant degradation by engineered microbiomes are promising and widely anticipated wastewater treatment approaches. There have been a few experimental studies on microalgae-bacteria symbiotic systems, which have demonstrated the economic and effectiveness of the system due to its green and sustainable treatment process. Existing research on microalgae-bacteria symbiotic systems in wastewater treatment mainly focuses on bioflocculation, immobilized culture, and membrane bioreactors, which have been shown to have good treatment efficiency. Microalgae-bacteria symbiotic systems mainly rely on mixed culture or mixed encapsulation of microorganisms. However, the urgent goals of carbon neutrality and sustainable development require new requirements for the wastewater treatment industry, especially in terms of efficient light harvesting and waste utilization. In order to achieve sustainable waste treatment and utilization, strengthening the synergistic effect of bacteria and algae and improving the light utilization of microorganisms still requires further research. The emerging photofluidic technology has the characteristics of efficient light manipulation and utilization, can precisely control the composition and dynamics of the bacterial community, and achieve enhanced light-matter interaction. In addition, it can be combined with existing equipment to meet large-scale industrial applications. Summary of the invention

[0004] In order to enhance the capture of photons of natural light by microbial systems and improve the light collection efficiency, thereby improving the photosynthesis efficiency of microorganisms; and realize the sustainable utilization of waste, the present invention provides a configurable spatialized bacteria-algae integrated biophotonic system, which has excellent performance in wastewater treatment efficiency and sustainable utilization of waste.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A configurable spatialized bacteria-algae integrated biophotonic system, the configurable spatialized bacteria-algae integrated biophotonic system is a gel sphere with a core-shell structure, the sphere diameter of the gel sphere is 110-120 μm; the core layer of the core-shell structure comprises Chlorella vulgaris and a first hydrophilic carrier, and the shell layer comprises Bacillus licheniformis and a second hydrophilic carrier; the ratio of the sphere diameter of the core layer to the width of the shell layer is (0.3-3.75):1; the width of the shell layer is the difference between the sphere diameter of the gel sphere and the sphere diameter of the core layer.

[0007] Preferably, the ratio of the spherical diameter of the core layer to the width of the shell layer is (0.46-2.22):1.

[0008] Further preferably, the ratio of the spherical diameter of the core layer to the width of the shell layer is (0.92-1.11):1.

[0009] More preferably, the ratio of the spherical diameter of the core layer to the width of the shell layer is 1:1.

[0010] Preferably, the first hydrophilic carrier is a hydrogel, or / and the second hydrophilic carrier is a hydrogel.

[0011] The present invention also provides a microfluidic droplet generator for preparing the configurable spatialized bacteria-algae integrated biophotonic system, comprising a lower cavity, a glass plate, a microfluidic droplet chip and an upper cavity arranged in sequence from bottom to top; the upper cavity is provided with a first inlet channel, a second inlet channel, a third inlet channel and an outlet channel; the microfluidic droplet chip is bonded to the glass plate by plasma; the lower cavity accommodates the glass plate and the microfluidic droplet chip; the upper cavity cover forms an integral structure on the lower cavity, namely the microfluidic droplet generator.

[0012] Preferably, the microfluidic droplet generator comprises a plurality of identical microfluidic droplet generating units, each of which comprises a first microfluidic channel, a second microfluidic channel, a third microfluidic channel, a fourth microfluidic channel and a fifth microfluidic channel; the first microfluidic channel and the second microfluidic channel are symmetrically arranged on both sides of the third microfluidic channel, and the ends of both are connected to the third microfluidic channel to form a first confluence point; the fourth microfluidic channel and the fifth microfluidic channel are symmetrically arranged on both sides of the third microfluidic channel, and the ends of both are connected to the third microfluidic channel to form a second confluence point; the second confluence point is downstream of the first confluence point; the first inlet channel is connected to the third microfluidic channel; the second inlet channel is connected to both the first microfluidic channel and the second microfluidic channel; the third inlet channel is connected to both the fourth microfluidic channel and the fifth microfluidic channel; the second confluence point is connected to the outlet channel.

[0013] The method for preparing the configurable spatialized bacteria-algae integrated biophotonic system using the microfluidic droplet generator comprises the following steps:

[0014] S1, preparing a reaction solution: preparing an aqueous phase I containing Chlorella and a substance capable of forming a first hydrophilic carrier, preparing an aqueous phase II containing Bacillus licheniformis and a substance capable of forming a second hydrophilic carrier, and preparing an oil phase containing a surfactant;

[0015] S2. The aqueous phase I is mixed with the aqueous phase II, and the obtained mixed liquid is sheared into micro-droplets by the oil phase; the micro-droplets react with a cross-linking agent to obtain a gel sphere, or the micro-droplets are photocured in the presence of an initiator to obtain a gel sphere, and the gel sphere is the configurable spatialized bacteria-algae integrated biophotonic system.

[0016] Preferably, the flow rate ratio of the aqueous phase I, the flow rate of the aqueous phase II and the flow rate of the oil phase is (1:3:16) to (3:1:16).

[0017] Further preferably, the flow rate ratio of the aqueous phase I, the flow rate of the aqueous phase II and the flow rate of the oil phase is (1:2:12.12) to (2:1:12.12).

[0018] More preferably, the flow rate ratio of the aqueous phase I, the flow rate of the aqueous phase II and the flow rate of the oil phase is 1:1:8.

[0019] Preferably, the crosslinking agent is Ca-containing 2+ or / and Sr 2+ of solution.

[0020] Preferably, the substance capable of forming the first hydrophilic carrier is sodium alginate, and the first hydrophilic carrier is a hydrogel formed by sodium alginate and the cross-linking agent.

[0021] Preferably, the substance capable of forming the second hydrophilic carrier is sodium alginate, and the second hydrophilic carrier is a hydrogel formed by sodium alginate and the cross-linking agent.

[0022] Preferably, the initiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

[0023] Preferably, the substance capable of forming the first hydrophilic carrier comprises at least one of methacryloyl gelatin, methacryloyl hyaluronic acid, polyethylene glycol, and chitosan. The first hydrophilic carrier comprises a hydrogel obtained by curing at least one of methacryloyl gelatin, methacryloyl hyaluronic acid, polyethylene glycol, and chitosan with visible light or ultraviolet light in the presence of the initiator.

[0024] Preferably, the substance capable of forming the second hydrophilic carrier comprises at least one of methacryloyl gelatin, methacryloyl hyaluronic acid, polyethylene glycol, and chitosan. The second hydrophilic carrier comprises a hydrogel obtained by curing at least one of methacryloyl gelatin, methacryloyl hyaluronic acid, polyethylene glycol, and chitosan with visible light or ultraviolet light in the presence of the initiator.

[0025] Preferably, the oil phase is liquid paraffin.

[0026] Preferably, the surfactant is Span 80.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) In the present invention, Chlorella and Bacillus licheniformis are prepared into transparent gel spheres, which improves the capture rate of natural photons by microorganisms, thereby improving the photosynthesis of microorganisms; and the metabolic waste produced by one microorganism becomes the nutrient of another microorganism, thereby improving the utilization rate of metabolic waste produced by microorganisms. It also increases the bioaccumulation of Chlorella and enhances the synergistic effect between microorganisms.

[0029] (2) When the configurable spatial bacteria-algae integrated biophoton system is used to treat wastewater, the degradation efficiency of total nitrogen, total phosphorus and chemical oxygen demand can reach 89.89%, 91.8% and 91.98% respectively within 72 hours. Compared with the co-culture group of Bacillus licheniformis and Chlorella vulgaris, the configurable spatial bacteria-algae integrated biophoton system in the present invention improves the degradation efficiency of TN, TP and COD by 63.7%, 75.6% and 73.4% respectively.

[0030] (3) Compared with the existing millimeter-level gel spheres, the micrometer-level configurable spatialized bacteria-algae integrated biophotonic system prepared in the present invention increases the blue light intensity by 106.09% and the red light intensity by 94.99%, and the utilization rate of light is greatly improved. This is because the microorganisms in the configurable spatialized bacteria-algae integrated biophotonic system have better dispersion and larger specific surface area, so the contact area with light and nitrogen and phosphorus in wastewater is larger, the utilization rate of light is higher, and the dissipation of light is greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the structure and working principle of the configurable spatial bacteria-algae integrated biophotonic system of the present invention and a comparison diagram of the light capture capability with the existing millimeter-scale immobilized gel beads;

[0032] Figure 2The microfluidic droplet generator structure diagram and the result diagram of the effect of the flow rate ratio of the center flow and the sheath flow on the core-shell structure size of the configurable spatial bacteria-algae integrated biophotonic system in the present invention;

[0033] Figure 3 This is a graph showing the degradation efficiency of TN, TP and COD by gel spheres of different size structures in Example 3;

[0034] Figure 4 The confocal fluorescence microscope imaging diagram of the configurable spatial bacteria-algae integrated biophotonic system in Example 3 and the result diagram of the optical density, lipid content and chlorophyll a content of the microbial system of each experimental group changing with the culture time;

[0035] Figure 5 The degradation effect diagram of the microbial system of each experimental group in Example 3 on total nitrogen, total phosphorus and COD respectively;

[0036] Figure 6 This is a comparison result of the light capture capability between the configurable spatially integrated bacteria-algae biophotonic system and the millimeter-scale immobilized gel beads in Example 3;

[0037] Figure 7 This is a comparison diagram of the degradation efficiency of TN, TP, and COD between the configurable spatially integrated bacteria-algae biophotonic system and the millimeter-scale gel beads in Example 3.

[0038] In the figure: 1. first inlet channel; 2. second inlet channel; 3. third inlet channel; 4. outlet channel; 5. lower cavity; 6. glass plate; 7. microfluidic droplet chip; 8. upper cavity; 9. first microfluidic channel; 10. second microfluidic channel; 11. third microfluidic channel; 12. fourth microfluidic channel; 13. fifth microfluidic channel. DETAILED DESCRIPTION

[0039] The following is a clear and complete description of the technical solution of the present invention in combination with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, any equivalent transformation or substitution made by ordinary technicians in this field according to the following implementation methods without creative work shall fall within the protection scope of the present invention.

[0040] The Chlorella vulgaris (FACHB-8) used in the following examples was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The main components of the BG-11 medium used to culture Chlorella cells are as follows: 1.76×10 -2 mol / L NaNO 3 , 2×10 - 4 mol / LK 2 HPO4 , 3×10 -4 mol / L MgSO 4 7H 2 O, 1×10 -4 mol / L CaCl 2 ·2H 2 O, 2×10 -4 mol / LNa 2 CO 3 , 3×10 -5 mol / L citric acid, 2×10 -5 mol / L ammonium ferric citrate, 3×10 -6 mol / L EDTA, 4.61×10 - 2 mol / LH 3 BO 3 , 9×10 -5 mol / L MnCl 2 ·4H 2 O, 8×10 -4 mol / L ZnSO 4 7H 2 O, 1.9×10 -3 mol / LNa 2 MoO 4 , 2×10 -4 mol / L CuSO 4 ·5H 2 O, 2×10 -4 mol / L Co(NO 3 ) 2 6H 2 O, 1 mL / L A5 trace element solution and 919 mL / L distilled water; culture medium pH = 7.0.

[0041] The culture method of Chlorella cells is as follows: Chlorella is cultured in 250 mL BG-11 medium, and irradiated alternately during the day (12 h, 3000 lux) and at night (12 h) in a light incubator (ZQLY-180GV) at 25°C. The optical density (OD) is measured at 680 nm using a UV-visible spectrometer (SPECORD 210PLUS, Analytikjena, Germany). 680nm ) to determine the number of cells.

[0042] The Bacillus licheniformis is deposited under the accession number CGMCC 1.7461 from the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences (Beijing, China). The main components of the Lysogeny Broth (LB) liquid medium used to culture Bacillus licheniformis cells are as follows: 10 g / L trypsin, 5 g / L yeast extract, 10 g / L sodium chloride; the medium pH = 7.0.

[0043] The culture method of Bacillus licheniformis is as follows: Bacillus licheniformis is cultured in 250 mL LB liquid medium, and irradiated alternately during the day (12 h, 3000 lux) and at night (12 h) in a light incubator (ZQLY-180GV) at 25°C. The optical density (OD) measured at 600 nm by a UV-visible spectrometer (SPECORD 210PLUS, Analytikjena, Germany) is 600nm ) to determine the number of cells.

[0044] Example 1

[0045] like Figure 2 As shown in Figure a, this embodiment provides a microfluidic droplet generator, including a lower cavity 5, a glass plate 6, a microfluidic droplet chip 7 and an upper cavity 8 arranged in sequence from bottom to top; wherein the upper cavity 8 is provided with a first inlet channel 1, a second inlet channel 2, a third inlet channel 3 and an outlet channel 4 in parallel. The microfluidic droplet chip 7 is bonded to the glass plate 6 by plasma bonding. The lower cavity 5 serves as a bottom supporting structure to accommodate the glass plate 6 and the microfluidic droplet chip 7. The upper cavity 8 covers the lower cavity 5 to form an integral structure.

[0046] like Figure 2 As shown in Figure b, the microfluidic droplet chip includes several microfluidic droplet generation units. Each of the microfluidic droplet generation units includes five microfluidic channels, namely the first microfluidic channel 9, the second microfluidic channel 10, the third microfluidic channel 11, the fourth microfluidic channel 12, and the fifth microfluidic channel 13. The inner diameters of the first microfluidic channel 9, the second microfluidic channel 10, the fourth microfluidic channel 12, and the fifth microfluidic channel 13 are all 116 μm; the inner diameter of the third microfluidic channel 11 is 250 μm. The first microfluidic channel 9 and the second microfluidic channel 10 are symmetrically arranged on both sides of the third microfluidic channel 11, and the ends of both are connected to the third microfluidic channel 11 to form a first confluence point; the fourth microfluidic channel 12 and the fifth microfluidic channel 13 are symmetrically arranged on both sides of the third microfluidic channel 11, and the ends of both are connected to the third microfluidic channel 11 to form a second confluence point; the second confluence point is downstream of the first confluence point.

[0047] like Figure 2 As shown in Figure c, there are three laminar flows in Figure c, of which two red laminar flows (i.e., sheath flows, respectively flowing in the first microfluidic channel 9 and the second microfluidic channel 10 in Figure b) are both solutions mixed with 1.5% (w / w) sodium alginate solution and rhodamine B; the green laminar flow (i.e., the center flow, flowing in the third microfluidic channel 11 in Figure b) is a solution mixed with 1.5% (w / w) sodium alginate solution and rhodamine 123; these three laminar flows form droplets under the action of the shear sheath flow at the junction. The microfluidic droplet generator is manufactured according to the soft lithography process commonly used in the art, and a homogenizer is used to spin-coat SU-8 2050 negative photoresist with a thickness of 100 μm on the wafer. After pre-baking, the wafer and the glass mask are exposed to ultraviolet light on a photolithography machine (e.g., H94-37 6" double-sided photolithography machine produced by Sichuan Nanguang Vacuum Technology Co., Ltd.). Then, after post-baking and development processes, a chemically and thermally stable photoresist image is obtained on the silicon wafer as a chip template. Polydimethylsiloxane (PDMS) is poured into a silicon wafer-based mold and baked in an oven (model: XMTD-8222) for 1 hour, and then the PDMS replica is peeled off and sealed on a flat glass to form a microchannel.

[0048] Example 2

[0049] This embodiment provides a method for preparing a configurable spatialized bacteria-algae integrated biophotonic system, comprising the following steps:

[0050] S1. Prepare reaction solution

[0051] Take 1 mL of the cultured Chlorella solution (OD 680nm =0.11) washed twice with 0.85wt% sodium chloride solution and centrifuged at 5000rpm for 5 minutes; after removing the supernatant, 1mL1.5% (w / w) sodium alginate solution was added to obtain aqueous phase I.

[0052] Take 1 mL of the cultured Bacillus licheniformis solution (OD 600nm =0.15) washed twice with 0.85wt% NaCl solution and centrifuged at 7000rpm for 10 minutes; after removing the supernatant, 1mL 1.5% (w / w) sodium alginate solution was added to obtain aqueous phase II.

[0053] Liquid paraffin oil supplemented with 1% (w / w) Span 80 surfactant was used as the oil phase.

[0054] S2. Preparation of configurable spatially integrated bacterial and algal biophotonic system

[0055] The aqueous phase I enters the microfluidic droplet generator through the first inlet channel 1 at a flow rate of 50 to 150 μL / h; the aqueous phase II enters the microfluidic droplet generator through the second inlet channel 2 at a flow rate of 50 to 150 μL / h; the oil phase enters the microfluidic droplet generator through the third inlet channel 3 at a flow rate of 800 μL / h; the generated sodium alginate droplets flow out of the outlet channel 4 and pass through a hose into a 2% (w / w) CaCl 2 In the solution, sodium alginate and Ca 2+ Cross-linking to produce a core-shell structured gel sphere is a configurable spatially integrated bacteria-algae biophotonic system.

[0056] like Figure 2 As shown in Figures b and c, the aqueous phase I and the aqueous phase II enter the microfluidic droplet generator from the first inlet channel 1 and the second inlet channel 2 respectively, and then enter each microfluidic droplet generation unit respectively; the oil phase enters the microfluidic droplet generator from the third inlet channel 3 and then enters each microfluidic droplet generation unit respectively. In each microfluidic droplet generation unit, the aqueous phase I flows in the third microfluidic channel 11 (i.e., the central flow), and the aqueous phase II flows in the first microfluidic channel 9 and the second microfluidic channel 10 (i.e., the sheath flow); the oil phase flows in the fourth microfluidic channel 12 and the fifth microfluidic channel 13, and then merges with the aqueous phase I and the aqueous phase II in the third microfluidic channel 11. The mixed liquid of aqueous phase I and aqueous phase II forms droplets at the junction of the fourth microfluidic channel 12, the fifth microfluidic channel 13 and the third microfluidic channel 11 under the action of the shear sheath flow of the oil phase. The droplets contain Chlorella, Bacillus licheniformis and sodium alginate, and the sodium alginate in the droplets is cross-linked with calcium ions to form a double-layer micron-sized gel sphere consisting of a core layer and a shell layer.

[0057] The results of the effect of liquid flow rate ratio on the structure of the configurable spatially integrated bacteria-algae biophotonic system are shown in Table 1.

[0058] Table 1 Effect of liquid flow rate ratio on the structure of configurable spatially integrated bacteria-algae biophotonic system

[0059]

[0060] Table 1 lists the ratio of the core diameter (Wc) to the shell width (Ws, the shell width is twice the shell thickness) of the obtained gel sphere (i.e., the configurable spatialized bacteria-algae integrated biophoton system) when the flow rate ratio of the water phase I, water phase II, and oil phase is (1:3:16) to (3:1:16) in the preparation process of the configurable spatialized bacteria-algae integrated biophoton system is (0.3 to 3.75):1; when the flow rate ratio of the water phase I, water phase II, and oil phase is (1:2:12.12) to (2:1:12.12), the ratio of the core diameter (Wc) to the shell width (Ws) of the obtained gel sphere is (0.46 to 2.22):1; when the flow rate ratio of the water phase I, water phase II, and oil phase is 1:1:8, the ratio of the core diameter (Wc) to the shell width (Ws) of the obtained gel sphere is (0.92 to 1.11):1. Figure 2 As shown in Figure d, the ratio of the core diameter to the shell width of the gel sphere increases as the ratio of the center flow velocity (Qc) to the sheath flow velocity (Qs) increases. Figure 2 As shown in Figure e, the spherical diameters of 226 gel spheres were measured at different flow rates of water phase I, water phase II and oil phase, and were all distributed between 110 and 120 μm.

[0061] It should be noted that the calcium chloride solution in this embodiment can also be replaced by other salt solutions that can form a transparent hydrogel with the sodium alginate solution, such as other solutions containing calcium ions or solutions containing strontium ions (Sr 2+ ) solution. The first hydrophilic carrier sodium alginate solution can also be replaced by other substances that can form transparent hydrogels, such as photosensitive hydrogels such as methacryloyl gelatin (GelMA), methacryloyl hyaluronic acid (HAMA), polyethylene glycol (PEG) and chitosan, etc. These hydrogels form transparent hydrogels after visible light or ultraviolet light curing in the presence of an initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP). Similarly, the second hydrophilic carrier sodium alginate solution can also be replaced by other substances that can form transparent hydrogels.

[0062] Example 3

[0063] This embodiment provides a method for treating wastewater by using a configurable spatialized bacteria-algae integrated biophotonic system. The gel spheres of different sizes and structures prepared in Example 2 are added to the wastewater, respectively, and cultured at 25°C for 72 hours at an oscillation speed of 120 rpm / min in a light incubator (ZQLY-180GV, Shanghai Zhichu Instrument Co., Ltd.), and irradiated alternately during the day (12 hours, 3000 lux) and at night (12 hours). After the culture, the experimental groups with gel spheres of different sizes are centrifuged at 12000 rpm for 10 minutes, and then the degradation of total nitrogen (TN), total phosphorus (TP) and chemical oxygen demand (COD) in the wastewater by gel spheres of various sizes is recorded every 12 hours.

[0064] Main components of wastewater: 300mg / L glucose, 22.16mg / L NH 4 Cl, 6.57 mg / L KNO 3 , 1.08mg / LNaNO 2 and 5.09mg / L KH 2 PO 3 The initial concentrations of TN, TP and COD in the wastewater were 30.0 mg / L, 5.0 mg / L and 171.338 mg / L, respectively (reference: "The interactions of algae-bacteria symbiotic system and its effects on nutrients removal from synthetic wastewater", DOI: https: / / doi.org / 10.1016 / j.biortech.2017.09.074).

[0065] 3.1 Determination of total nitrogen (TN) content

[0066] The total nitrogen content was determined according to the APHA method (Standard Methods for the Examination of Water and Wastewater: American Public Health Association, Washington, DC, USA. 2012). The color of the TN content was characterized by colorimetry using the Japanese KYORITSU water quality kit total nitrogen kit, and the operation method was carried out according to the instructions of the kit. Yellow corresponds to 0 mg / L TN content, lime green corresponds to 5 mg / L TN content, light green corresponds to 10 mg / L TN content, green corresponds to 25 mg / L TN content, cyan corresponds to 50 mg / L TN content, and blue-gray corresponds to 100 mg / L TN content.

[0067] 3.2 Determination of total phosphorus (TP) content

[0068] The total phosphorus content was determined according to the APHA method (Standard Methods for the Examination of Water and Wastewater: American Public Health Association, Washington, DC, USA. 2012). The color of the TP content was characterized using the Japanese KYORITSU water quality kit total phosphorus kit, and the operation method was carried out according to the instructions of the kit. White corresponds to a TP content of 0.2 mg / L, aqua corresponds to a TP content of 0.5 mg / L, light blue-green corresponds to a TP content of 1 mg / L, sky blue corresponds to a TP content of 2 mg / L, blue corresponds to a TP content of 5 mg / L, and dark blue corresponds to a TP content of 10 mg / L.

[0069] 3.3 Chemical oxygen demand (COD) determination

[0070] COD content is measured according to APHA method (Standard Methods for the Examination of Water and Wastewater: American Public Health Association, Washington, DC, USA. 2012). The color of COD content presented by the Japanese KYORITSU water quality kit chemical oxygen demand kit is characterized by colorimetry, and the operation method is carried out according to the instructions of the kit. Purple corresponds to 0 mg / L COD content, dark purple corresponds to 30 mg / L COD content, dark cyan corresponds to 60 mg / L COD content, green corresponds to 120 mg / L COD content, olive corresponds to 200 mg / L COD content, and brown corresponds to more than 250 mg / L COD content.

[0071] Reference Figure 3 The degradation efficiencies of gel spheres with different sizes and structures on TN, TP and COD are as follows: Figure 3 As shown in Figures a, b, and c, the dotted lines on the "bottles" of various shapes in the figure represent the median degradation efficiency. Figure 3It can be seen that the median degradation efficiency of gel spheres of different sizes (Wc / Ws is (0.3-3.75):1) prepared in Example 2 for TN, TP and COD can reach more than 87%, and the degradation efficiency increases first and then decreases with the increase of the Wc / Ws ratio; when the Wc / Ws ratio is (0.46-2.22):1, the median degradation efficiency of TN reaches 89.15%, the median degradation efficiency of TP reaches 90%, and the median degradation efficiency of COD reaches 90.4%. When the Wc / Ws ratio is (0.92-1.11):1, the degradation efficiency reaches the maximum, the median degradation efficiency of TN reaches 90.5%, the median degradation efficiency of TP reaches 93.6%, and the median degradation efficiency of COD reaches 92.7%.

[0072] The gel sphere with a Wc / Ws ratio of 1:1 prepared in the embodiment (i.e., a spatialized bacteria-algae integrated biophotonic system) was selected for control experiments with other microbial systems. The wastewater used was the same as the wastewater in steps 3.1 to 3.3. The methods for treating wastewater by the microbial systems in each experimental group and the methods for determining TN, TP, and COD were the same as those in steps 3.1 to 3.3. The optical density, lipid content, chlorophyll a content, and the degradation of total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (COD) of each experimental group were determined every 12 hours according to the methods in steps 3.4 to 3.6.

[0073] Among them, the four different microbial systems are as follows:

[0074] (1) Chlorella group: initial OD 680nm =0.11;

[0075] (2) Bacillus licheniformis group: initial OD 600nm =0.15;

[0076] (3) Bacillus licheniformis and Chlorella co-culture group (also called non-immobilized bacteria-algae co-culture): initial OD 600nm =0.15, initial OD 680nm =0.11;

[0077] (4) Configurable spatially integrated bacterial and algal biophotonic system: initial OD 600nm =0.15, initial OD 680nm =0.11, more than 3 million units.

[0078] 3.4 The optical density measurement method is as follows:

[0079] After the co-cultivation of the microbial system and wastewater, the Chlorella group, the Bacillus licheniformis group, and the Bacillus licheniformis and Chlorella co-cultivation group were washed twice with distilled water, and each was centrifuged at 7000 rpm for 5 minutes; after removing the supernatant, the precipitate of each group was resuspended in distilled water. The optical density (OD) of Chlorella was measured at 680 nm using a UV-visible spectrometer (SPECORD210PLUS). 680nm ), and the optical density (OD 600nm ).

[0080] The microbial system of group (4) was dissolved with 2% (w / v) sodium carbonate solution (Sigma-Aldrich, USA), and then washed twice with distilled water. The optical density (OD) of Chlorella vulgaris was measured at 680 nm using a UV-visible spectrometer (SPECORD 210PLUS). 680nm ), and the optical density (OD 600nm ). Distilled water was used as blank control.

[0081] 3.5 The lipid content was determined as follows:

[0082] After the co-cultivation of the microbial system and wastewater, the Chlorella cell pellets of each experimental group were centrifuged at 12000rpm for 10 minutes, the supernatant was discarded and resuspended in a mixture of chloroform and methanol (volume ratio of 2:1), and then stored in a mixture of chloroform and methanol prepared at a volume ratio of 2:1 at 4°C for 12 hours. Take it out and put it in an ultrasonic cleaner (model: KQ-300DE, Kunshan Shumei) for 10 minutes. The freshly extracted lipids were centrifuged again at 12000rpm for 10 minutes to obtain a transparent supernatant, and the solvent was removed by vacuum evaporation; after drying under nitrogen, the lipids were weighed to quantify the lipid content of each experimental group.

[0083] 3.6 The determination method of chlorophyll a content is as follows:

[0084] After the co-cultivation of the microbial system and wastewater, the microbial system of each experimental group was centrifuged at 12000rpm for 10 minutes, the supernatant was removed, and the microbial system was resuspended in 90% acetone solution and treated with an ultrasonic cleaner (model: KQ-300DE, Kunshan Shumei) for 10 minutes to extract chlorophyll a. The absorbance was measured at 645nm and 663nm using a spectrophotometer, and the 90% acetone solution was used as a blank control to determine the chlorophyll a content in the extracted supernatant. Chlorophyll a (C a The concentration (mg / L) of ) was calculated by the following formula: (C a =12.7×OD 663nm -2.69×OD 645nm)(Wang H, Qi B, Jiang 663nm Indicates the absorbance measured at 663nm, OD 645nm It represents the absorbance measured at 645 nm.

[0085] The configurable spatialized bacteria-algae integrated biophotonic system was imaged using a confocal fluorescence microscope at the end of the culture at 72 hours. Bacillus licheniformis was labeled with the green fluorescent dye Meilun green (Dalian Meilun Biotechnology Co., Ltd.), and Chlorella was labeled with Nile red. The imaging results are shown in Figure 4 As shown in Figure a; the fluorescence intensity analysis results are shown in Figure 4 As shown in Figure b; the results show that during the processing process, the configurable spatially integrated bacteria-algae biophotonic system maintained good stability and microbial encapsulation. Figure 4 Figure c shows the optical density (OD) of Chlorella in each experimental group. 680nm ) determination results, and Figure d shows the optical density (OD 600nm ), Figure e is the determination results of lipid content in each experimental group, and Figure f is the determination results of chlorophyll a content in each experimental group.

[0086] The results of TN degradation in each experimental group are shown in Figure 5 Figure a and b. Figure 5 As can be seen from Figure a, the degradation efficiencies of TN within 72 hours by the Chlorella group, the Bacillus licheniformis and Chlorella co-culture group, and the configurable spatially integrated bacteria-algae biophotonic system were 35.61%, 72.19%, and 89.89%, respectively. Figure 5 As can be seen from Figure b, after the configurable spatially integrated bacteria-algae biophotonic system was co-cultured with wastewater for 72 hours, the colorimetric result of TN detection in the wastewater was yellow; this shows that among all the experimental groups, the configurable spatially integrated bacteria-algae biophotonic system had the best nitrogen degradation effect.

[0087] The results of TP degradation in each experimental group are shown in Figure 5 Figures c and d in the middle. Figure 5 As can be seen from Figure c, the degradation efficiencies of TP in the Chlorella group, the Bacillus licheniformis and Chlorella co-culture group, and the configurable spatially integrated bacteria-algae biophotonic system were 47.44%, 66.36%, and 91.8%, respectively, within 72 hours. Figure 5 As can be seen from Figure d, after the configurable spatially integrated bacteria-algae biophotonic system was co-cultured with the wastewater for 72 hours, the colorimetric result of TP detection in the wastewater was white; this shows that among all the experimental groups, the configurable spatially integrated bacteria-algae biophotonic system had the best effect on phosphorus degradation.

[0088] The results of COD degradation in each experimental group are shown in Figure 5 Figures e and f in the middle. Figure 5 As can be seen from Figure e, the COD degradation efficiencies of the Chlorella group, the Bacillus licheniformis and Chlorella co-culture group, and the configurable spatial bacteria-algae integrated biophotonic system within 72 hours were 40.51%, 69.87%, and 91.98%, respectively; Figure 5 As can be seen from Figure f, after the configurable spatially integrated bacteria-algae biophotonic system was co-cultured with the wastewater for 72 hours, the colorimetric result of COD detection in the wastewater was purple; this shows that among all the experimental groups, the configurable spatially integrated bacteria-algae biophotonic system had the best COD degradation effect.

[0089] right Figure 4 and Figure 5 The measurement results were statistically analyzed and the data processing results are shown in Table 2.

[0090] Table 2 Statistical analysis results of various indicators of each group of microbial systems

[0091]

[0092]

[0093] From the OD in Table 2 680nm From the data of OD, lipid content data, and chlorophyll a content data, it can be seen that among all experimental groups, the configurable spatial bacteria-algae integrated biophotonic system performed best in accumulating Chlorella biomass. 600nm The data show that compared with other experimental groups, the configurable space-based bacteria-algae integrated biophoton system has a significant advantage in the growth of Bacillus licheniformis. From the data of TN, TP and COD, it can be seen that in each experimental group, the configurable space-based bacteria-algae integrated biophoton system has the best effect on the degradation of wastewater. Compared with the co-culture group of Bacillus licheniformis and Chlorella, the configurable space-based bacteria-algae integrated biophoton system in the present invention has increased the degradation efficiency of TN, TP and COD by 63.7%, 75.6% and 73.4%, respectively.

[0094] The statistical and analytical results of various aspects show that the configurable space-based bacteria-algae integrated biophotonic system has excellent degradation performance in wastewater treatment. This is because the light-collecting capacity of the configurable space-based bacteria-algae integrated biophotonic system is enhanced, and the Bacillus licheniformis in its shell oxidizes the organic matter in the wastewater and decomposes it into ammonia nitrogen (NH 3 -N), phosphate and carbon dioxide, these nutrients together with light provide nutrition and energy source for the core part of Chlorella, which can synthesize biomass and release oxygen through photosynthesis; then Bacillus licheniformis continues to oxidize organic matter with oxygen, thus forming a carbon cycle (such as Figure 1 (As shown in Figure a). On the other hand, in the configurable spatial bacteria-algae integrated biophotonic system, Chlorella and Bacillus licheniformis were effectively dispersed, thereby obtaining more growth space and a larger specific surface area, thereby increasing the contact area between microorganisms and nitrogen, phosphorus and light, and enhancing the ability to capture photons. However, in the co-culture group of Bacillus licheniformis and Chlorella, Chlorella and Bacillus licheniformis tend to grow in clusters during suspension culture, resulting in a dispersion that is not as good as the configurable spatial bacteria-algae integrated biophotonic system, which is not conducive to improving the efficiency of light utilization. Figure 1 As shown in Figures b and c, the shell layer of the configurable spatially integrated bacteria-algae biophotonic system in Figure b is marked with 1μm red polystyrene fluorescent microspheres, and the core layer is marked with 5μm green polystyrene fluorescent microspheres. Figure c is the light intensity analysis of Figure b. It can be seen from Figures b and c that the Chlorella is obviously dispersed more evenly.

[0095] like Figure 1As shown in Figures d to f, Figure d is a comparison of the light capture capabilities of the existing immobilized millimeter-scale gel beads and the configurable spatialized bacteria-algae integrated biophoton system, Figure e is the regionalized red, green and blue light grayscale values ​​of the existing immobilized millimeter-scale gel beads, and Figure f is the regionalized red, green and blue light grayscale values ​​of the configurable spatialized bacteria-algae integrated biophoton system. It can be seen from Figures d to f that the light capture capability of the configurable spatialized bacteria-algae integrated biophoton system is significantly stronger than that of the existing immobilized millimeter-scale gel beads. This is mainly because the light-collecting capability of the configurable spatialized bacteria-algae integrated biophoton system is enhanced in two ways: first, the ability to adjust the capture of natural light photons is optimized, and second, the light attenuation of the system is directly reduced. The means of optimizing the capture of natural light photons can be achieved by adjusting the size of the configurable spatialized bacteria-algae integrated biophoton system to enhance the interaction between light and matter. Since the configurable spatialized bacteria-algae integrated biophotonic system has good dispersion and increases the specific surface area of ​​microorganisms, light and microorganisms can fully contact and reduce light dissipation defined in the Bouguer-Lambert-Beer law. The 100-micron size of the configurable spatialized bacteria-algae integrated biophotonic system can not only ensure the enhanced modulation optimization of natural light transmission, but also encapsulate sufficient biomass.

[0096] Figure 6 By analyzing the light intensity, the existing mainstream immobilized millimeter-scale gel beads and the configurable spatially integrated bacteria-algae biophotonic system were compared at the same Chlorella concentration (OD 680nm =0.3). Light intensity is an important parameter reflecting the photosynthetic efficiency of Chlorella. Many studies have shown that blue light and red light are beneficial to the growth of Chlorella. The current mainstream method for preparing millimeter-scale immobilized gel beads is as follows: Mix the aqueous phase I and aqueous phase II prepared in step S1 of Example 2 evenly, draw 1 mL of the mixed solution with a syringe, and drip it into 2% (w / w) CaCl 2 Millimeter-sized immobilized gel beads are formed in the solution. Figure 6 As shown, Figure 6Figure a in the middle shows the maximum blue regionalized grayscale values ​​of the configurable spatialized bacteria-algae integrated biophotonic system and the millimeter-scale immobilized gel beads, which are 36.18 (SD: 3.059) and 13.62 (SD: 2.456), respectively; Figure b shows the maximum red regionalized grayscale values ​​of the configurable spatialized bacteria-algae integrated biophotonic system and the millimeter-scale immobilized gel beads, which are 36.29 (SD: 2.808) and 14.48 (SD: 1.868), respectively; Figure c shows the maximum green regionalized grayscale values ​​of the configurable spatialized bacteria-algae integrated biophotonic system and the millimeter-scale immobilized gel beads, which are 35.08 (SD: 3.832) and 14.61 (SD: 2.278), respectively. ; Figure d shows the average blue regionalized grayscale values ​​of the configurable spatialized bacteria-algae integrated biophotonic system and the millimeter-scale immobilized gel beads, which are 21.19 (SD: 2.913) and 10.28 (SD: 1.604), respectively; Figure e shows the average red regionalized grayscale values ​​of the configurable spatialized bacteria-algae integrated biophotonic system and the millimeter-scale immobilized gel beads, which are 20.25 (SD: 2.169) and 10.39 (SD: 1.469), respectively; Figure f shows the average green regionalized grayscale values ​​of the configurable spatialized bacteria-algae integrated biophotonic system and the millimeter-scale immobilized gel beads, which are 20.62 (SD: 2.705) and 10.6 (SD: 1.035), respectively. That is, Figures a to f show the transmittance of the configurable spatialized bacteria-algae integrated biophotonic system and the millimeter-scale immobilized gel beads based on surface domain image analysis. From Figure 6 It can be seen that there is a significant difference in light intensity between the configurable spatial bacteria-algae integrated biophoton system and the mainstream immobilized millimeter-scale gel beads. Compared with the mainstream immobilized millimeter-scale gel beads, the configurable spatial bacteria-algae integrated biophoton system increased the blue light intensity by 106.09% and the red light intensity by 94.99%. This enhancement in light collection should be attributed to the hundred-micron-level optical system, which can greatly reduce light dissipation and promote light transmission. These statistical results prove that the light collection capacity of the configurable spatial bacteria-algae integrated biophoton system has been significantly enhanced.

[0097] Figure 7 Figures a, b, and c are respectively the comparison results of the degradation rates of TN, TP, and COD of the configurable spatialized bacteria-algae integrated microbial photon system prepared in this application and the existing millimeter-scale immobilized gel beads. It can be seen from the figure that although the existing millimeter-scale immobilized gel beads are also gel spheres with Chlorella as the core layer and Bacillus licheniformis as the shell layer, their degradation efficiency of TN, TP, and COD is less than 80%. This shows that the improvement of the utilization efficiency of light by the configurable spatialized bacteria-algae integrated biophoton system prepared in this application is significantly helpful to improve the wastewater treatment capacity.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For any person skilled in the art, the present invention may have various changes and modifications. Any simple equivalent changes and modifications made according to the scope of protection of the present invention and the contents of the specification should be included in the scope of protection of the present invention.

Claims

1. A configurable spatial bacteria-algae integrated biophotonic system, characterized in that: The configurable spatialized bacteria-algae integrated biophotonic system is a transparent gel sphere with a core-shell structure, the core layer diameter of the gel sphere is 55-60 μm; the core layer of the core-shell structure contains Chlorella and a first hydrophilic carrier, and the shell layer contains Bacillus licheniformis and a second hydrophilic carrier; the ratio of the diameter of the core layer to the width of the shell layer is 1:1; the width of the shell layer is the difference between the diameter of the gel sphere and the diameter of the core layer; the first hydrophilic carrier and the second hydrophilic carrier are both hydrogels formed by sodium alginate and a cross-linking agent.

2. The method for preparing the configurable spatialized bacteria-algae integrated biophotonic system according to claim 1, characterized in that: The configurable spatialized bacteria-algae integrated biophotonic system is prepared by using a microfluidic droplet generator, comprising the following steps: S1, preparing a reaction solution: preparing an aqueous phase I containing Chlorella and a substance capable of forming a first hydrophilic carrier, preparing an aqueous phase II containing Bacillus licheniformis and a substance capable of forming a second hydrophilic carrier, and preparing an oil phase containing a surfactant; the substance capable of forming the first hydrophilic carrier is sodium alginate, and the substance capable of forming the second hydrophilic carrier is sodium alginate; S2, mixing the aqueous phase I and the aqueous phase II, and the obtained mixed liquid is sheared into microdroplets by the oil phase; the microdroplets react with a cross-linking agent to obtain a gel sphere, and the gel sphere is the configurable spatialized bacteria-algae integrated biophotonic system; the flow rate of the aqueous phase I, the flow rate of the aqueous phase II and the flow rate of the oil phase are in a ratio of 1:1:

8.

3. The method for preparing the configurable spatialized bacteria-algae integrated biophotonic system according to claim 2, characterized in that: The crosslinking agent is Ca-containing 2+ or / and Sr 2+ of solution.

4. The method for preparing a configurable spatially integrated bacteria-algae biophotonic system according to claim 2, characterized in that: The oil phase is liquid paraffin.

5. The method for preparing a configurable spatialized bacteria-algae integrated biophotonic system according to claim 2, characterized in that: The surfactant is Span 80.

6. Application of the configurable spatialized bacteria-algae integrated biophotonic system according to claim 1 in treating wastewater.

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