Lignin degradation complexing agent and application thereof in papermaking wastewater treatment

Abstract

The invention discloses a lignin degradation complexing agent. The lignin degradation complexing agent is prepared from haematococcus pluvialis and bacteria TD18, wherein the cell concentration of the haematococcus pluvialis is 5 * 10 < 5 > cell * mL <-1 >, and the cell concentration of the bacteria TD18 is 5 * 10 < 6 > cell * mL <-1 >; the method comprises the following steps: preparing a haematococcus pluvialis seed solution and a bacteria TD18 seed solution, and then mixing the haematococcus pluvialis seed solution and the bacteria TD18 seed solution according to a cell quantity ratio of 10: 1-10: 6, and inoculating the mixture into sewage containing lignin. The complexing agent can tolerate lignin and can synergistically and efficiently remove lignin and nitrogen in sewage, the sewage treatment effect is good, by means of the lignin degradation complexing agent, the removal rate of the lignin in the sewage is 8.42%-100%, and the removal rate of the lignin is 5.61 mg.( L.d) <-1 >-41.67 mg.( L.d) <-1 >; meanwhile, the culture method is simple, low in cost and suitable for preparing products for sewage treatment.

Description

Technical Field

[0001] The invention relates to the field of sewage treatment by microorganisms, and in particular to a lignin degradation composite agent and application thereof in papermaking wastewater treatment. Background Art

[0002] Papermaking wastewater is a typical organic wastewater with high pollution, high oxygen consumption and difficulty in biodegradation. The discharge volume and main pollutants of papermaking wastewater, such as lignin, vary due to different papermaking raw materials and papermaking processes. The lignin content in papermaking wastewater is very high and is the main source of water pollution. Therefore, finding a comprehensive treatment technology for papermaking wastewater that meets national conditions will be of great significance to protecting the environment and developing my country's papermaking industry.

[0003] Traditional methods for treating papermaking wastewater include physical and chemical methods, chemical methods and biological methods. Physical and chemical methods include chemical oxidation-adsorption method, coagulation method, extraction method, electrochemical method and incineration method, etc. Chemical methods include ozone oxidation method, oxidation method, electrochemical oxidation method, etc. The biological method mainly uses microorganisms to oxidize, degrade and adsorb organic pollutants in wastewater, and use these substances as their own nutrients, while purifying the wastewater. The physical materials (such as extractants) and chemical reagents used in the treatment of papermaking wastewater by physical and chemical methods are expensive and may also cause secondary pollution to the environment. Compared with the two, the advantages of biological treatment of papermaking wastewater are low operating costs, simple operation and low secondary pollution. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a lignin degradation composite agent and its application in papermaking wastewater treatment.

[0005] In order to solve the above technical problems, the present invention proposes a lignin degradation composite agent, which is composed of Haematococcus pluvialis and bacteria TD18; wherein the cell concentration of Haematococcus pluvialis is 5×10 5 Cells·mL -1 The cell concentration of bacterial TD18 was 5×10 6 Cells·mL -1 ; and prepared according to the following method:

[0006] Preparation of Haematococcus pluvialis seed solution: After obtaining the culture solution of Haematococcus pluvialis, inoculate it into sterile BBM medium, culture it in a light incubator, collect the cells by centrifugation, and dilute it with sterile BBM medium to obtain a cell concentration of 5×10 5 Cells·mL -1 of Haematococcus pluvialis seed liquid;

[0007] Preparation of bacterial TD18 seed solution: streak the refrigerated bacterial TD18 on solid LB medium, culture and activate it in an incubator, then ferment the activated bacterial TD18 in liquid LB medium in a light-controlled constant temperature shaker to obtain bacterial fermentation broth, centrifuge, collect cells, and dilute with sterile BBM medium to obtain a cell concentration of 5×10 6 Cells·mL -1 of bacterial TD18 seed liquid.

[0008] Furthermore, after preparing the Haematococcus pluvialis seed solution and the bacterial TD18 seed solution, the Haematococcus pluvialis seed solution and the bacterial TD18 seed solution are mixed according to a cell number ratio of 10:1 to 10:6.

[0009] The above lignin degradation composite agent is used in the treatment of papermaking wastewater, wherein the wastewater contains lignin and nitrate nitrogen, and Haematococcus pluvialis seed liquid and bacteria TD18 seed liquid are inoculated into the wastewater respectively, and a light intensity of 60 μmol / (m 2 ·s).

[0010] Furthermore, in the above application, the lignin concentration in the sewage is 0-500 mg·L -1 , and is not equal to 0; the Haematococcus pluvialis seed liquid and sewage are inoculated at a volume ratio of 1:10; the bacteria TD18 seed liquid and sewage are inoculated at a volume ratio of 1:1000 to 6:1000); the inoculation ratio of Haematococcus pluvialis and bacteria TD18 is 10:1, to 10:6.

[0011] The lignin degradation composite agent of the present invention is used in the treatment of papermaking wastewater, and the lignin removal rate is 8.42% to 100%, and the lignin removal rate is 5.61 to 41.67 mg·(L·d) -1 .

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention provides a composite microbial agent composed of Haematococcus pluvialis and bacteria TD18, wherein both the algae and the bacteria can process lignin, and can effectively remove lignin and total nitrogen under the optimal inoculation ratio. The composite microbial agent can be applied to papermaking wastewater treatment, has good sewage treatment effect and strong adaptability, and the cultivation method is simple and the cost is low, and is suitable for preparing sewage treatment products. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure shows the removal of lignin from papermaking wastewater with different concentrations by Haematococcus pluvialis and bacteria TD18 under separate culture conditions.

[0015] Figure 2 The results are as follows: the inoculation ratios of Haematococcus pluvialis and bacteria TD18 were different for 400 mg·L -1 Removal of lignin from papermaking effluent.

[0016] Figure 3 The figure shows the removal of lignin in high-concentration papermaking wastewater by Haematococcus pluvialis and bacteria TD18 at the optimal inoculation ratio.

[0017] Figure 4 The figure shows the removal of nitrate nitrogen in high-concentration papermaking wastewater by Haematococcus pluvialis and bacteria TD18 at the optimal inoculation ratio. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the following embodiments are by no means intended to limit the present invention in any way.

[0019] The culture medium used in the following examples was prepared as follows:

[0020] BBM (Bold's Basal Medium) culture medium, add the following ingredients to every 1L of distilled water: 250mgNaNO3, 175mg KH2PO4, 75mg MgSO4·7H2O, 75mg K2HPO4, 50mg EDTA·Na2, 31mg KOH, 25mgNaCl, 25mg CaCl2·2H2O, 11.4mg H3BO3, 8.82mg ZnSO4·7H2O, 1.44mg MnCl2·4H2O, 1.79mg Na2MoO4, 1.57mg CuSO4·5H2O, 0.49mg Co(NO3)2·6H2O, 4.98mg FeSO4·7H2O and 1mLH2SO4.

[0021] LB (Luria-Bertani) medium is divided into solid LB medium and liquid LB medium. Among them, LB solid medium is to add the following ingredients to 1L of distilled water: 5g yeast extract, 10g peptone, 10g NaCl and 18g agar. Compared with solid LB medium, liquid LB medium does not need to add agar.

[0022] Example 1

[0023] Preparation of lignin degradation compound and papermaking wastewater and lignin detection method

[0024] 1) Preparation of a lignin degradation compound, comprising:

[0025] Preparation of Haematococcus pluvialis seed solution: 10 mL of Haematococcus pluvialis culture solution was inoculated into a 250 mL conical flask containing 100 mL of sterile BBM medium and placed in a light incubator (25 °C, light intensity 60 μmol / (m 2 ·s)) and after reaching a stable growth period (generally 7 days), 8000r·min -1 The cells were collected by centrifugation for 10 min and diluted with sterile BBM medium to obtain a cell concentration of 5 × 10 5 Cells·mL -1 of Haematococcus pluvialis seed liquid.

[0026] Preparation of bacterial TD18 seed solution: streak the refrigerated bacterial TD18 on solid LB medium, incubate in an incubator (25°C) for 48 h to activate the strain, scrape the activated bacterial TD18 with an inoculation loop and place in 100 mL of liquid LB medium, place in a light-controlled constant temperature shaker (25°C, light intensity 60 μmol / (m 2 ·s), 120r·min -1 ,) and fermented for 24 h. The bacterial fermentation broth was centrifuged (12000 r / min, 10 min), the cells were collected, and diluted with sterile BBM medium to obtain a cell concentration of 5×10 6 Cells·mL -1 of bacterial TD18 seed liquid.

[0027] 2) Simulate papermaking wastewater containing lignin in the laboratory: Take 1g of alkaline lignin, add BBM culture medium, make up to 100mL volumetric flask, and sterilize to obtain 10g / L lignin mother solution. Determination of lignin concentration: Use lignin as solute and BBM culture medium as solvent to prepare a lignin solution with a mass concentration of 1g / L, perform full-band scanning at a wavelength of 220-650nm, and determine that the optimal absorption wavelength of alkaline lignin is 234nm. Prepare alkaline lignin standard solutions with concentrations of 20, 40, 60, 80, and 100mg / L respectively, measure the absorbance of the standard solution at 234nm, draw a standard curve with alkaline lignin mass concentration (X) as the horizontal coordinate and absorbance (Y) as the vertical coordinate, and get the regression equation after fitting: y=0.01402x-0.02767, (R 2 =0.99053).

[0028] In the present invention, Haematococcus pluvialis seed liquid and bacteria TD18 seed liquid (the cell number is the same as that of Haematococcus pluvialis) are inoculated separately or simultaneously into papermaking wastewater of different concentrations to obtain culture solution, 5 mL of the culture solution is centrifuged, 3 mL of the supernatant is taken, the absorbance of the supernatant at 234 nm is measured, and the mass concentration of alkaline lignin is calculated by the regression equation of the alkaline lignin standard curve.

[0029] Example 2

[0030] Treatment of papermaking wastewater with different concentrations by separate cultivation of Haematococcus pluvialis and bacteria TD18

[0031] The seed solution of Haematococcus pluvialis (10% inoculation volume ratio) and the corresponding volume of bacterial TD18 seed solution (the cell number is the same as that of Haematococcus pluvialis) were inoculated into 250 mL conical flasks containing 100 mL of papermaking wastewater with different concentrations (the concentration of lignin was 0 mg·L -1 、100mg·L -1 , 200mg·L -1 、300mg·L -1 , 400mg·L -1 , 500mg·L -1 The conical flasks used in the above inoculation experiments were placed in a light incubator (25°C, light intensity 60 μmol / (m 2 ·s)) and were cultured statically, shaken twice a day, and each group of experiments was repeated three times. After 12 days of culture, the lignin concentration (mg·L -1 ), and based on this, the lignin removal rate (%) is calculated, and the calculation method is shown in formula (1):

[0032] N=(N0-N t ) / N0×100% (1)

[0033] In formula (1), N is the removal rate of lignin, %; N0 and N t are the lignin in the solution at day t0 and day t n Mass concentration per day, mg·L -1 .

[0034] like Figure 1 As shown in the figure, it was calculated that Haematococcus pluvialis and bacteria TD18 were cultured alone for 12 days to 100-300 mg·L -1 The removal rate of lignin was 100%, and the removal rate of lignin was 100% for 400 and 500 mg·L -1 The removal rates of lignin were 27.6% and 3.15% and 61.47% and 20.83% respectively, which indicated that both Haematococcus pluvialis and bacteria TD18 could utilize and metabolize lignin, and then remove lignin from papermaking wastewater.

[0035] Example 3

[0036] Determination of the optimal inoculation ratio of mixed culture of Haematococcus pluvialis and bacteria TD18

[0037] The experiment in Example 2 found that the lowest concentration of lignin that could not be completely removed by Haematococcus pluvialis and bacteria TD18 was 400 mg·L -1 Therefore, when the lignin concentration is 400 mg·L -1Mixed culture of algae and bacteria was carried out at the same time to determine the optimal inoculation ratio of mixed culture of Haematococcus pluvialis and bacteria TD18.

[0038] Four samples were prepared: 10 mL (10% inoculation amount) of Haematococcus pluvialis was inoculated into 100 mL of 400 mg·L -1 The corresponding volume of bacterial TD18 seed solution was inoculated into 250 mL conical flasks of papermaking wastewater at the same time, so that the cell number ratio of Haematococcus pluvialis to bacterial TD18 reached 10:1, 10:2, 10:4 and 10:6, respectively. Haematococcus pluvialis (10 mL) was cultured alone (CH) and bacterial TD18 (1 mL) was cultured alone (CG) as controls. The conical flasks for the above inoculation experiments were placed in a light incubator (25 ° C, light intensity 60 μmol / (m 2 ·s)) and were cultured statically, shaken twice a day, and each group of experiments was repeated three times. After 12 days of culture, the lignin concentration (mg·L -1 ), and based on this, the lignin removal rate (%) and removal rate (mg·(L·d) -1 ), the lignin removal rate was calculated according to formula (1), and the lignin removal rate was calculated according to formula (2).

[0039] R=(N0-N t ) / (t n -t0) (2)

[0040] In formula (2), R is the removal rate of lignin, mg·(L·d) -1 ; N0 and N t are the lignin in the solution at day t0 and day t n Mass concentration per day, mg·L -1 .

[0041] like Figure 2 As shown, four mixed culture systems (M 10 / 1 、M 10 / 2 、M 10 / 4 and M 10 / 6 ) had a higher lignin removal rate and removal rate than CH (27.6%, 9.2 mg·(L·d) -1 ) and CG (61.47%, 20.49 mg·(L·d) -1 ), where M 10 / 4 The removal rate of the culture system was the highest (100%), and the removal rate reached the maximum value of 41.3 mg·(L·d) -1 , followed by M 10 / 6 (91.06%, 30.35 mg·(L·d) -1 ), M 10 / 2 (87.9%, 29.3 mg·(L·d) -1), M 10 / 1 (79.42%, 26.47mg·(L·d) -1 ). This indicates that there is a synergistic effect between Haematococcus pluvialis and bacteria TD18, and the optimal cell number ratio of Haematococcus pluvialis and bacteria TD18 mixed culture is 10:4, at which the lignin removal efficiency is the highest.

[0042] Example 4

[0043] Treatment of high-concentration lignin in papermaking wastewater by Haematococcus pluvialis and bacteria TD18 at the optimal inoculation ratio

[0044] The experiment in Example 3 found that the lignin removal effect of Haematococcus pluvialis and bacteria TD18 was the best at the optimal inoculation ratio of 10:4. Therefore, Haematococcus pluvialis and bacteria TD18 were inoculated in a 250 mL conical flask containing 100 mL of high-concentration papermaking wastewater at a cell number ratio of 10:4 (lignin concentrations were 400 mg·L -1 (M400), 500mg·L -1 (M500), 600mg·L -1 (M600), 700mg·L -1 (M700) and 800mg·L -1 The conical flasks used in the above inoculation experiments were placed in a light incubator (25°C, light intensity (cloudy) 60 μmol / (m 2 ·s)) and were cultured statically, shaken twice a day, and each group of experiments was repeated three times. After 12 days of culture, the lignin concentration (mg·L -1 ), and based on this, the lignin removal rate (%) and removal rate (mg·(L·d) -1 ), the removal efficiency and removal rate of lignin were calculated according to formula (1) and formula (2), respectively.

[0045] like Figure 3 As shown, M 400 、M 500 、M 600 、M 700 and M 800 The removal rates of medium-alkaline lignin were 100% (10d), 100%, 83.18%, 37.65% and 8.42% (12d), respectively, and the removal rates were 40 mg·(L·d) -1 、41.67mg·(L·d) -1 、41.64mg·(L·d) -1 、21.96mg·(L·d) -1 and 5.61mg·(L·d) -1 . Compared with single culture (such as Figure 1Compared with the above results, the optimal inoculation ratio of 10:4 can further improve the removal rate of lignin, especially the complete removal of 500 mg·L -1 Alkaline lignin, M 500 Removal rate of medium alkaline lignin (41.67 mg·(L·d) -1 ) is Haematococcus pluvialis (1.31 mg·(L·d) -1 ) and bacteria TD18 (8.68 mg·(L·d) -1 ) were 31.75 and 4.8 times of those cultured alone. This indicates that Haematococcus pluvialis and bacteria TD18 can treat papermaking wastewater with a higher concentration at a cell number ratio of 10:4.

[0046] Example 5

[0047] Treatment of high-concentration nitrate nitrogen in papermaking wastewater by Haematococcus pluvialis and bacteria TD18 at the optimal inoculation ratio

[0048] As in Example 4, after 12 days of cultivation, the nitrate nitrogen concentrations (mg·L) in M400, M500, M600, M700 and M800 were measured. -1 ).

[0049] like Figure 4 As shown, M 400 and M 500 The nitrate nitrogen in the culture system was completely removed on the 10th and 12th days. On the 12th day, M 600 、M 700 and M 800 The nitrate nitrogen content in the culture system was 11.5 mg·L -1 、123.93mg·L -1 and 173.4 mg·L -1 This indicates that Haematococcus pluvialis and bacteria TD18 can efficiently remove high-concentration nitrate nitrogen from papermaking wastewater at a cell number ratio of 10:4.

[0050] Although the present invention has been described above in conjunction with the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can make many improvements and changes without departing from the purpose of the present invention, which are all within the protection of the present invention.

Claims

1. A lignin degradation composite agent, characterized in that: The method is composed of Haematococcus pluvialis and bacteria TD18; wherein the cell concentration of Haematococcus pluvialis is 5×10 5 Cells·mL -1 The cell concentration of bacterial TD18 was 5×10 6 Cells·mL -1 ; and prepared according to the following steps: Step 1) Preparation of Haematococcus pluvialis seed solution: After obtaining the Haematococcus pluvialis culture solution, inoculate it into a sterile BBM medium, culture it in a light incubator, collect the cells by centrifugation, and dilute it with a sterile BBM medium to obtain a cell concentration of 5×10 5 Cells·mL -1 of Haematococcus pluvialis seed liquid; Step 2) Preparation of bacterial TD18 seed solution: streaking the refrigerated bacterial TD18 on solid LB medium, culturing and activating in an incubator, and then fermenting the activated bacterial TD18 in liquid LB medium in a light-controlled constant temperature shaker to obtain bacterial fermentation broth, centrifuging, collecting cells, and diluting with sterile BBM medium to obtain a cell concentration of 5×10 6 Cells·mL -1 of bacterial TD18 seed liquid.

2. The lignin degradation composite agent according to claim 1, characterized in that: After preparing the Haematococcus pluvialis seed solution and the bacterial TD18 seed solution, the Haematococcus pluvialis seed solution and the bacterial TD18 seed solution are mixed according to a cell number ratio of 10:1 to 10:

6.

3. The use of the lignin degradation composite agent according to claim 1 in papermaking wastewater treatment, characterized in that: The sewage is sewage containing lignin and nitric nitrogen. The seed liquid of Haematococcus pluvialis and the seed liquid of bacteria TD18 are mixed and inoculated into the sewage. At room temperature, a light intensity of 60 μmol / (m 2 ·s).

4. The use of the lignin degradation composite agent according to claim 3 in papermaking wastewater treatment, characterized in that: The lignin concentration in the sewage is 400-800 mg·L -1 , and is not equal to 0; the Haematococcus pluvialis seed liquid and sewage are inoculated at a volume ratio of 1:10; the bacteria TD18 seed liquid and sewage are inoculated at a volume ratio of 1:1000 to 6:1000; the inoculation ratio of Haematococcus pluvialis and bacteria TD18 is 10:1 to 10:

6.

5. The use of the lignin degradation composite agent according to claim 4 in papermaking wastewater treatment, characterized in that: The lignin removal rate is 8.42% to 100%, and the lignin removal rate is 5.61 to 41.67 mg·(L·d) -1 .

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