WO2019041792A1 - 一种强化人工湿地去除水体微囊藻及毒素的方法及系统 - Google Patents

一种强化人工湿地去除水体微囊藻及毒素的方法及系统 Download PDF

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WO2019041792A1
WO2019041792A1 PCT/CN2018/080411 CN2018080411W WO2019041792A1 WO 2019041792 A1 WO2019041792 A1 WO 2019041792A1 CN 2018080411 W CN2018080411 W CN 2018080411W WO 2019041792 A1 WO2019041792 A1 WO 2019041792A1
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wetland
microcystins
water
layer
degrading bacteria
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PCT/CN2018/080411
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English (en)
French (fr)
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邰义萍
万翔
杨扬
满滢
王瑞
王佳希
杨瑜芬
段杰瀚
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暨南大学
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

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  • the invention relates to the technical field of water treatment, and more particularly to a method for enhancing the removal of water-producing algae and microcystins by artificial wetland, which is suitable for use as a drinking water source or an eutrophic water body, microcystis and toxins, Easy handling.
  • Toxic cyanobacterial blooms not only seriously damage the aquatic ecosystem, but also release algal toxins, which threaten the health of aquatic plants and humans, among which Microcystins (microcystins) MCs) is the most common cyanobacterial toxin that causes the most serious damage.
  • Microcystin is generally produced and present in algae cells, but under certain conditions, algae cells will rupture and intracellular algal toxins (IMC) will be released into the water body.
  • IMC intracellular algal toxins
  • EMC dissolved extracellular toxin
  • microcystin has been found in more than 90
  • the most common and most influential configurations are MC-LR, MC-RR and MC-YR.
  • the persistence of algal toxins increases the removal load of sewage treatment plants and also causes the drinking risk of tap water plants. Long-term drinking of algae-containing water can cause liver cancer and kidney damage.
  • the treatment methods for algae mainly include artificial fishing, flocculation, chemical method, air flotation and chemical oxidation.
  • the method for removing microcystins can be roughly divided into physical method, chemical method and biological method according to the removal principle. 3 categories, more common such as air floatation, activated carbon adsorption, ozone oxidation, Fenton oxidation. Most of the physical methods remove microcystis cells to remove intracellular algal toxins (IMC), but the removal of extracellular toxins (EMC) in water is not ideal, and it is easy to break algae cells and increase the cells in the water. Exotoxin.
  • IMC intracellular algal toxins
  • EMC extracellular toxins
  • the chemical method mainly destroys the structure of MCs by oxidation, and most of them have good treatment effects on intracellular and extracellular toxins, but the large amount of chemical agents are easy to produce toxic and harmful by-products, causing secondary pollution and processing costs. Extremely high, difficult to apply on a large scale.
  • the object of the present invention is to overcome the deficiencies of the prior art processing techniques and to provide a method for enhancing the removal of microcystis and toxins from aquatic bodies by using exogenous microcystins degrading bacteria.
  • Another technical problem to be solved by the present invention is to provide a constructed wetland system that implements the method of the present invention.
  • the present invention adopts the following technical solutions:
  • the method of applying exogenous microcystin-degrading bacteria is to enrich and increase the concentration of microcystins from the water of the microcystis bloom, and periodically detect the degradation gene mlrA during the enrichment process to confirm the culture time of the degrading bacteria.
  • the culture solution is applied to a constructed wetland after the concentration of the microcystins-degrading bacteria in the microcystins-degrading bacterial liquid is 1 ⁇ 10 6 to 2 ⁇ 10 6 cells/m 2 or more.
  • the artificial wetland adopts a submerged artificial wetland, and the wetland matrix is divided into three layers, the bottom layer, the middle layer and the surface layer from bottom to top, the bottom layer is zeolite, the middle layer is gravel, and the surface layer is high organic matter peat soil. Plant perennial aquatic herbs.
  • the method for applying the exogenous microcystins-degrading bacteria uniformly spreads the laboratory-accumulated and domesticated microcystin-degrading bacteria at a concentration of 1 ⁇ 10 6 to 2 ⁇ 10 6 cells/m 2 or more to the artificial
  • the surface of the wetland is ploughed and used after being vacant.
  • the vacant time is about one day.
  • the method further comprises feeding the microcystins-containing sewage into the constructed wetland. It is preferably input through a drip irrigation device.
  • the underlayer has a thickness of 5 to 9 cm; further preferably, the zeolite has a particle diameter of 1 to 2 cm.
  • the intermediate layer has a thickness of 15 to 20 cm; further preferably, the crushed stone has a particle diameter of 0.5 to 1 cm.
  • the surface layer has a thickness of 5 to 10 cm.
  • the use of high organic peat soil on the surface layer is conducive to microbial fixation and reproduction.
  • the perennial aquatic herb is yellow calamus or the like.
  • the method comprises the steps of:
  • Eutrophic lake water film treatment Before the operation of the constructed wetland system, the wetland is hanged by eutrophic lake water to enrich the indigenous microorganisms in the wetland and accelerate the formation of biofilm, which is conducive to the fixation of exogenous degrading bacteria;
  • the high algae toxin load sewage drip irrigation method is:
  • the enriched and domesticated microcystin-degrading bacterial liquid is uniformly sprinkled on the surface layer of the constructed wetland substrate at a concentration of 1 ⁇ 10 6 to 2 ⁇ 10 6 cells/m 2 or more, and is ploughed, and vacant for use ( grow plants);
  • the low algae toxin load sewage drip irrigation method is
  • the microcystin-degrading bacterial liquid after acclimation in step S4 is cultured with LB medium (composition: peptone 5.0 g, yeast extract 2.5 g, NaCl 5.0 g, sterilized ultrapure water 500 mL, adjusted pH 7.0), and used After the physiological saline is resuspended, it is used for spraying.
  • LB medium composition: peptone 5.0 g, yeast extract 2.5 g, NaCl 5.0 g, sterilized ultrapure water 500 mL, adjusted pH 7.0
  • the invention also provides a constructed wetland system for realizing the method of the invention, comprising a submerged flow constructed wetland, a microcystins degrading bacterial liquid transport device and a transport device containing microcystins sewage, the submerged artificial wetland comprising a wetland main body, a water device and a drainage device, wherein the wetland body is filled with a wetland substrate, the water inlet device is disposed on the side wall of the wetland body, and the drainage device is disposed at the bottom of the wetland body; and the water inlet device is respectively connected to the conveying device containing the microcystins sewage And a transport device for microcystin-degrading bacterial liquid.
  • the system further comprises an effluent sampling device and a sewage retention treatment device, the effluent sampling device being connected to the drainage device, the sewage retention treatment device being connected to the drainage device at one end and the wetland body at the other end.
  • the water inlet device adopts a drip irrigation method.
  • the wetland matrix is divided into three layers, the bottom layer, the middle layer and the surface layer from bottom to top, the bottom layer is zeolite, the middle layer is gravel, the surface layer is high organic matter peat soil, and perennial aquatic herb plants are planted.
  • the underlayer has a thickness of 5 to 9 cm; further preferably, the zeolite has a particle diameter of 1 to 2 cm.
  • the intermediate layer has a thickness of 15 to 20 cm; further preferably, the crushed stone has a particle diameter of 0.5 to 1 cm.
  • the surface layer has a thickness of 5 to 10 cm.
  • the use of high organic peat soil on the surface layer is conducive to microbial fixation and reproduction.
  • the perennial aquatic herb is yellow calamus or the like.
  • the wetland body is a cylindrical structure.
  • the cylindrical structure has a diameter of 22 cm and a height of 35 cm, and the total height of the wet substrate is 30 cm.
  • the invention has the following beneficial effects:
  • the applicant overcomes the defects and limitations of the prior art, and combines the interaction of exogenous microcystins-degrading bacteria and artificial wetland biofilm indigenous microorganisms with the advantages of constructed wetlands to thereby construct artificial wetlands.
  • the treatment technology is creatively applied to remove microcystis and toxins from water bodies, and to take advantage of ecological treatment technology, without causing secondary pollution, low operating cost, rapid treatment, and good removal potential for extracellular toxins and intracellular toxins. It can be well connected with existing water treatment technology and has a good application prospect.
  • the method of the invention has low operating cost and high efficiency, and the total nitrogen removal rate is over 67.5%, the ammonia nitrogen removal rate is over 52.6%, the nitrate nitrogen removal rate is over 91.6%, and the total phosphorus removal rate is up to More than 76.0%, the cell-removing rate of the toxin-producing algae is as high as 95.4%, and the microcystins removal rate is as high as 98.4%, which effectively reduces the ecological risk of algae toxin caused by cyanobacterial blooms.
  • the microcystins degrading bacterial liquid is treated, and the matrix in the strong wetland system exerts a synergistic effect of physical, chemical and biological, further detoxifying the microcystins, and accelerating the treatment of microcystins by the wetland.
  • the effect is to reduce the residence time of the water in the wetland to treat the algal toxin, improve the removal load of the algae toxin in the wetland per unit time, and remove the probiotic algae cells and toxins.
  • the removal rate of the toxin-producing algae cells is as high as 95.4%, Microcystis
  • the toxin removal rate is as high as 98.4%.
  • microcystins The treatment of microcystins is flexible, and the reaction time and the amount of exogenous degrading bacteria can be flexibly selected according to the residual algal toxin content in the water body, and the utility model has strong practicability and can be well interfaced with the existing water treatment process.
  • the method of the invention is economical and feasible, does not require additional chemical reagents, and the required material price is low and common, and does not significantly increase the cost of water treatment, and the filler replacement is simpler and does not cause secondary pollution.
  • the method of the present invention further optimizes the constructed wetland system to better implement the present invention.
  • Figure 1 is a schematic flow diagram of the method of the present invention.
  • Fig. 2 is a schematic view showing the structure of the system for removing microcystis and toxins from water bodies.
  • 11 is a water inlet device
  • 121 is a substrate surface layer
  • 122 is a matrix intermediate layer
  • 123 is a matrix bottom layer
  • 14 is an aquatic plant
  • 15 is a drainage device (drainage port)
  • 2 is a microcystins-degrading bacterial liquid delivery device.
  • 3 is a conveying device containing microcystins sewage.
  • Figure 3 is an electrophoresis map of the exogenous microcystin-degrading bacteria gene mlrA.
  • Fig. 4 is a schematic diagram showing changes in microcystin in water treated by constructed wetland.
  • the present embodiment provides a method for enhancing the removal of microcystis and toxins from water bodies by using artificial wetlands, and applying the interaction of exogenous microcystins-degrading bacteria and artificial microorganisms of constructed wetlands to improve the removal efficiency of algae and toxins in constructed wetlands.
  • the method for applying the exogenous microcystins degrading bacteria is to enrich and increase the concentration of microcystins from the water body of the microcystis bloom, and periodically detect the degrading bacteria gene mlrA during the enrichment process to confirm the degrading bacteria
  • the culture time is adjusted until the concentration of the microcystins-degrading bacteria in the microcystins-degrading bacterial solution is 1 ⁇ 10 6 to 2 ⁇ 10 6 cells/m 2 or more, and the bacterial liquid is applied to the constructed wetland.
  • the constructed wetland adopts a submerged artificial wetland.
  • the wetland matrix is divided into three layers.
  • the bottom layer is the bottom layer, the middle layer and the surface layer.
  • the bottom layer is zeolite, the middle layer is gravel, the surface layer is high organic matter peat soil, and the perennial aquatic plant is planted. herb.
  • Eutrophic lake water film treatment Before the operation of the constructed wetland system, the wetland is hanged by eutrophic lake water to enrich the indigenous microorganisms in the wetland and accelerate the formation of biofilm, which is conducive to the fixation of exogenous degrading bacteria;
  • the high algae toxin load sewage drip irrigation method is:
  • the enriched and domesticated microcystin-degrading bacterial liquid is uniformly sprinkled on the surface layer of the constructed wetland substrate at a concentration of 1 ⁇ 10 6 to 2 ⁇ 10 6 cells/m 2 or more, and is ploughed, and vacant for use ( grow plants);
  • the low algae toxin load sewage drip irrigation method is
  • microcystins-degrading bacterial solution after acclimation in step S4 is cultured with LB medium (composition: protein gluten 5.0 g, yeast extract 2.5 g, NaCl 5.0 g, sterilized ultrapure water 500 mL, adjusted pH 7.0), and re-salted with physiological saline. After suspension, it is used for spraying.
  • LB medium composition: protein gluten 5.0 g, yeast extract 2.5 g, NaCl 5.0 g, sterilized ultrapure water 500 mL, adjusted pH 7.0
  • colony sequencing collecting surface water samples of long-term outbreaks of microcystis blooms and lakes, The DNA Isolation Kit (MO-BIO, USA) extraction kit extracts water-like DNA and then performs PCR amplification.
  • the DNA Isolation Kit MO-BIO, USA
  • the conditions for amplification were as follows: (1) The reaction system (25 ⁇ L) included 2.0 ⁇ L of DNA template, 2.5 ⁇ L of 10 ⁇ PCR buffer, 2.0 ⁇ L of MgCl 2 (25 ⁇ M), 2.0 ⁇ L of dNTPs (2.5 mM), PCR positive and negative primers ( 0.5 ⁇ L each of 10 ⁇ mol/L, 0.2 ⁇ L of Tap DNA polymerase (5 U/ ⁇ L), and supplemented with sterile deionized water. The primer information used is shown in Table 1.
  • Reaction system PCR amplification was carried out by a 96-well C100TM thermal cycler PCR machine (Bio-Rad, USA). PCR reaction conditions: (1) 95 ° C for 3 min; (2) 95 ° C for 30 s, 62 ° C for 1 min, 72 ° C for 1 min, 35 cycles; (3) 72 ° C for 7 min, 4 ° C forever.
  • the enrichment and culture operation of the bacterial liquid is as follows: after confirming the presence of microcystins-degrading bacteria in the bacterial source, the laboratory-extracted crude microcystins are used as the sole carbon and nitrogen source for enrichment, and the concentration of microcystins is gradually increased.
  • the degrading bacteria gene mlrA is periodically detected during the enrichment process to confirm that the degrading bacteria can be cultured.
  • the specific steps are as follows: First, a small amount of water from the lake is inoculated into the LB medium for 1 to 2 days, and then 1.0 mL of the enriched bacteria solution is inoculated into the inorganic salt medium containing the laboratory-extracted microcystins ( Composition: CuCl 2 , FeSO 4 , ZnCl 2 , MnCl 2 each 0.0005 g, CaCl 2 0.02 g, MgSO 4 ⁇ 7H 2 0 1.0 g, NaCl 1.0 g, KH 2 PO 4 0.5 g, K 2 HPO 4 4.0 g, The bacteria were ultra-pure water 1000 mL, the pH was adjusted to 7.5), and the temperature was oscillated (120 r/min, 30 ° C).
  • This embodiment also provides a preferred constructed wetland system, as shown in FIG.
  • the invention comprises a subsurface flow constructed wetland, a microcystin-degrading bacterial liquid transport device 2 and a transport device 3 containing microcystins sewage, the wet flow artificial wetland comprising a wetland main body, a water inlet device 11 and a drainage device 15, wherein the wetland body is The wetland substrate is loaded, and the water inlet device 11 is disposed on the side wall of the wetland body, preferably at the top of the side wall.
  • the drain device 15 is disposed at the bottom of the wetland main body; the water inlet device is respectively connected to the transport device 3 containing the microcystins sewage and the transport device 2 for the microcystins-degrading bacterial liquid.
  • the system further includes an effluent sampling device and a sewage septic treatment device, the effluent sampling device being connected to the drainage device, the sewage retention treatment device being connected to the drainage device at one end and the wetland body (not shown in Fig. 2) at the other end.
  • the water inlet device 11 adopts a drip irrigation method.
  • the wetland matrix is divided into three layers.
  • the bottom layer is the surface layer 121, the intermediate layer 122 and the bottom layer 123.
  • the bottom layer 123 is zeolite, the middle layer 122 is gravel, the surface layer 121 is high organic matter peat soil, and the perennial aquatic herb is planted. Plant 14.
  • the bottom layer 123 has a thickness of 5 to 9 cm; and the zeolite has a particle diameter of 1 to 2 cm.
  • the intermediate layer 122 has a thickness of 15 to 20 cm; and the crushed gravel has a particle diameter of 0.5 to 1 cm.
  • the surface layer has a thickness of 5 to 10 cm.
  • the use of high organic peat soil on the surface layer is conducive to microbial fixation and reproduction.
  • the perennial aquatic herb is yellow calamus and the like.
  • the wetland body may have a cylindrical structure.
  • Example 2 Test of removing nutrients such as nitrogen and phosphorus in the present invention
  • Example 2 Constructing and utilizing the submerged flow constructed wetland system and method described in Example 1, constructing an artificial wetland with an effective volume of 2.5L by using a 10 L PVC plastic bucket, the filling substrate is 5 cm soil layer from top to bottom, and 15 cm gravel (particle size 5 mm). Layer, the gravel was rinsed with tap water before filling, and the bottom layer was filled with 5 cm large particle zeolite (20 mm) to prevent water clogging, and 3 strains of yellow calamus were evenly planted in each device.
  • the eutrophic lake water is used for irrigation for 1 year.
  • the nutrient salt is added artificially, and the concentration of nutrients such as nitrogen and phosphorus in the influent water is shown in Table 2:
  • Nitrogen and phosphorus removal effect The constructed constructed wetland system has a stable effect on nitrogen and phosphorus nutrient removal.
  • the average removal rate of system TN is 67.5% ⁇ 75.3%
  • the average removal rate of NH 4 + -N is 52.6% ⁇ 66.3%
  • the average removal rate of NO 3 - -N is 91.6% ⁇ 94.2%
  • the average removal rate of TP It is 76.0% to 92.8%.
  • Example 3 Enhanced removal of algae cells and soluble microcystins LR by constructed wetlands
  • Example 2 The underflow artificial wetland system and method described in Example 1 were constructed and utilized, and the degrading bacteria composite bacterial liquid was evenly inverted on the surface soil of the wetland, and the composite bacterial liquid and the soil substrate were uniformly mixed by the tillage method, and domesticated for one week.
  • the initial microcystin MC-LR concentrations were set to 3.61 ⁇ g/L, 6.80 ⁇ g/L, and 16.07 ⁇ g/L, respectively.
  • After one-time influent stay in the wetland for 7 days, and collect 1 mL of water samples at 0h, 6h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 168h for determination of EMC-LR.
  • the optimal hydraulic retention time of the wetland system is 3d. Under this hydraulic retention time, the removal rate of the toxin-producing algae cells (intracellular toxin IMC-LR) by the constructed wetland system is 73.5%-95.4%, for extracellular microcystis The removal rate of toxin (EMC-LR) is 87.5% ⁇ 98.4%, which can effectively reduce the MC-LR content to below 1.0 ⁇ g/L of drinking water safety standard; further extend the hydraulic retention time of wetland system and enhance the removal effect of MC-LR. To the detection limit (0.10 ⁇ g/L).
  • microcystin complex degrading bacteria can significantly improve the removal rate of EMC-LR in wetland system (P ⁇ 0.05) and shorten the half-life to 22h, indicating that bio-enhanced constructed wetland can more effectively deal with microcystins pollution.
  • the present invention provides a method for using the exogenous microcystin-degrading bacteria to strengthen the artificial wetland to remove water microcystis and toxins, through indigenous microorganisms and exogenous addition in wetlands.
  • the biodegradation of microcystins-degrading bacteria enhances the harmless treatment of microcystins in wetland systems, and shows the advantages of simple operation, economical, rapid, efficient, flexible and no secondary pollution.
  • the invention is practical and adaptable to drinking water.
  • the processing needs of the source and eutrophic water can be well interfaced with existing water treatment processes.

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Abstract

一种强化人工湿地去除水体微囊藻及毒素的方法，其通过外源降解菌液的投加，增强湿地系统中基质发挥物理、化学和生物的协同作用，进一步对微囊藻毒素进行无害化处理。还公开了一种实现强化人工湿地去除水体微囊藻及毒素的方法的系统。

Description

一种强化人工湿地去除水体微囊藻及毒素的方法及系统 技术领域
本发明涉及水处理技术领域，更具体地，涉及一种强化人工湿地去除水体产毒藻及微囊藻毒素的处理方法，适合作为饮用水源或富营养化水体微囊藻及毒素的高效、简易处理。
背景技术
富营养化和气候变化引起的有毒蓝藻水华问题日益突出，有毒蓝藻水华不仅严重破坏水体生态系统，而且会释放藻毒素威胁水生动植物及人类的健康，其中微囊藻毒素(Microcystins，简称MCs)是最普遍，造成危害最为严重的一种蓝藻毒素，微囊藻毒素一般产生并存在于藻细胞体内，但在一定条件下，藻细胞会破裂，胞内藻毒素(IMC)会释放到水体中，成为溶解态的胞外毒素(EMC)，在藻华水体中普遍存在，并已在各种地表水体尤其是饮用水源水中常有检出，目前，微囊藻毒素已发现超过90多种构型，最常见也是影响最大的是MC-LR、MC-RR和MC-YR这三种。藻毒素的持续存在增加了污水处理厂的去除负荷，也引起自来水厂出水的饮用风险，长期饮用含藻毒素的水会引发肝癌、肾损伤等症状。
目前，针对藻类的处理方法主要包括人工捕捞、絮凝、化学药剂法、气浮和化学氧化法等，针对微囊藻毒素的去除方法按其去除原理可大致分为物理法、化学法、生物法3大类，比较常见如气浮、活性炭吸附、臭氧氧化、Fenton氧化等。物理法大部分通过去除微囊藻细胞从而去除胞内藻毒素(IMC)，但对水体中的胞外毒素(EMC)的去除效果并不理想，且容易使藻细胞破裂，增加水体中的胞外毒素。化学方法主要通过氧化作用破坏MCs的结构，大部分对胞内、胞外毒素均有较好的处理效果，但化学药剂的大量使用容易产生有毒有害副产物，带来二次污染，且处理成本极高，难以大规模应用。
目前未见将人工湿地处理技术应用于去除水体微囊藻及毒素的相关技术报道。
发明内容
本发明的目的是克服现有处理技术的不足，提供一种利用外源微囊藻毒素降解菌强化人工湿地去除水体微囊藻及毒素的方法。
本发明要解决的另一技术问题是提供实现本发明方法的人工湿地系统。
为解决上述技术问题，本发明采用以下技术方案：
提供一种强化人工湿地去除水体微囊藻及毒素的方法，是应用外源微囊藻毒素降解菌和人工湿地生物膜土著微生物的共同作用提高人工湿地对藻类及其毒素的去除效率；所述应用外源 微囊藻毒素降解菌方法是从爆发微囊藻水华的水体中富集并提高微囊藻毒素的浓度，在富集过程中定期检测降解菌基因mlrA，确认降解菌的培养时间，培养至微囊藻毒素降解菌液中微囊藻毒素降解菌的浓度为1×10 6～2×10 6细胞/m 2以上后将所述菌液应用于人工湿地。
优选地，所述人工湿地采用潜流人工湿地，湿地基质分为三层结构，自下而上依次为底层、中间层和表层，底层为沸石，中间层为碎砾石，表层为高有机质泥炭土，种植多年生水生草本植物。
优选地，所述应用外源微囊藻毒素降解菌方法将实验室富集、驯化的微囊藻毒素降解菌以1×10 6～2×10 6细胞/m 2以上的浓度均匀撒至人工湿地的表层，并翻耕，空置后使用。优选空置的时间为1天左右。
优选地，所述方法还包括将含微囊藻毒素的污水输入人工湿地。优选通过滴灌装置输入。
优选地，所述底层厚度为5～9cm；进一步优选所述沸石的粒径为1～2cm。优选地，所述中间层的厚度为15～20cm；进一步优选所述碎砾石的粒径为0.5～1cm。
优选地，所述表层的厚度为5～10cm。表层采用高有机质泥炭土有利于微生物固定和繁殖。
优选地，所述多年生水生草本植物为黄菖蒲等。
进一步优选地，所述方法包括以下步骤：
S1.构建人工湿地系统；所述系统的基质三层结构，自下而上依次为底层、中间层和表层，底层为沸石，中间层为碎砾石，表层为高有机质泥炭土，种植覆盖多年生水生草本植物；
S2.富营养化湖水挂膜处理：人工湿地系统运行前，通过富营养化湖水对湿地进行挂膜，以富集湿地中土著微生物，加速生物膜的形成，有利于外源降解菌的固定；
S3.高、低藻毒素负荷污水滴灌：
所述高藻毒素负荷污水滴灌方法是：
S31.从长期爆发微囊藻水华的水体筛选微囊藻毒素降解菌，以微囊藻毒素作为唯一碳氮源进行富集，并逐步提高微囊藻毒素的浓度；在富集过程中定期检测测降解菌基因mlrA，以确认降解菌的培养时间；
S32.将富集、驯化的微囊藻毒素降解菌液以1×10 6～2×10 6细胞/m 2以上的浓度均匀撒至人工湿地基质的表层上，并翻耕，空置后备用(种植植物)；
所述低藻毒素负荷污水滴灌方法是
S33.将含微囊藻毒素的污水输入人工湿地系统，停留一段时间，与湿地基质充分接触后作为 出水排出人工湿地系统；
S4.定时取出水样，分析其中营养盐浓度、微囊藻细胞数及毒素含量；如果未达标，将出水停留处理后返回步骤S3；达标则进行排放。
优选地，步骤S4驯化之后的微囊藻毒素降解菌液用LB培养基(组成：蛋白胨5.0g，酵母浸出液2.5g，NaCl 5.0g，灭菌超纯水500mL，调节pH为7.0)培养，并用生理盐水重新悬浮后用于投加喷撒。
本发明同时提供一种实现本发明方法的人工湿地系统，包括潜流人工湿地、微囊藻毒素降解菌液输送装置和含微囊藻毒素污水的输送装置，所述潜流人工湿地包括湿地主体、进水装置和排水装置，所述湿地主体内装填湿地基质，进水装置设置于湿地主体侧壁，排水装置设置于湿地主体的底部；所述进水装置分别连接含微囊藻毒素污水的输送装置和微囊藻毒素降解菌液的输送装置。
优选地，所述系统还包括出水取样装置和污水停留处理装置，所述出水取样装置连接排水装置，所述污水停留处理装置一端连接排水装置，另一端连接湿地主体。
优选地，所述进水装置采用滴灌方式。
优选地，所述湿地基质分为三层结构，自下而上依次为底层、中间层和表层，底层为沸石，中间层为碎砾石，表层为高有机质泥炭土，种植多年生水生草本植物。
优选地，所述底层厚度为5～9cm；进一步优选所述沸石的粒径为1～2cm。优选地，所述中间层的厚度为15～20cm；进一步优选所述碎砾石的粒径为0.5～1cm。
优选地，所述表层的厚度为5～10cm。表层采用高有机质泥炭土有利于微生物固定和繁殖。
优选地，所述多年生水生草本植物为黄菖蒲等。
优选地，所述湿地主体为圆柱形结构。
进一步优选地，所述圆柱形结构的直径为22cm，高度为35cm，湿地基质总高度为30cm。
本发明与现有技术相比，具有以下有益效果：
本申请人经过长期大量的研究总结，克服现有技术的缺陷和局限，将外源微囊藻毒素降解菌和人工湿地生物膜土著微生物的共同作用与人工湿地的优势有机结合，从而将人工湿地处理技术创造性应用于去除水体微囊藻及毒素，发挥生态处理技术的优势，不造成二次污染、运行成本低、处理快速、且对胞外毒素和胞内毒素均有较好的去除潜力，可与现有水处理技术 良好对接，有着很好的应用前景。
经过大量试验证明，本发明方法运行成本低、处理高效，对总氮去除率达67.5％以上，氨态氮去除率达52.6％以上，硝态氮去除率达91.6％以上，总磷去除率达76.0％以上，产毒藻细胞去除率高达95.4％，微囊藻毒素去除率高达98.4％，有效降低因蓝藻水华造成的水体藻毒素的生态风险。
本发明方法中外加微囊藻毒素降解菌液处理，强湿地系统中基质发挥物理、化学和生物的协同作用，进一步对微囊藻毒素进行无害化处理，可以加速湿地处理微囊藻毒素的效果，减少湿地处理藻毒素的水体停留时间，提高了单位时间内湿地处理藻毒素的去除负荷，对产毒藻细胞及毒素的去除效果好，产毒藻细胞去除率高达95.4％，微囊藻毒素去除率高达98.4％。处理微囊藻毒素操作灵活，可以根据水体中残留藻毒素含量，灵活选择反应时间和外源降解菌的添加量，实用性强，可以很好地与现有的水处理工艺对接。
本发明方法经济可行，不需外加化学试剂，所需材料价格较低且普遍，不会明显增加水处理的成本，填料更换更为简便，且不会造成二次污染。
本发明方法对人工湿地系统进行了进一步的优化，以更好地实现本发明。
附图说明
图1为本发明方法的流程示意图。
图2本发明去除水体微囊藻及毒素的系统结构示意图。
其中，11为进水装置，121为基质表层，122为基质中间层，123为基质底层，14为水生植物，15为排水装置(排水口)，2为微囊藻毒素降解菌液输送装置，3为含微囊藻毒素污水的输送装置。
图3为外源微囊藻毒素降解菌基因mlrA电泳图。
图4为人工湿地处理水体微囊藻毒素的变化示意图。
具体实施方式：
下面结合具体实施例进一步说明本发明。下述实施例仅用于示例性说明，不能理解为对本发明的限制。除非特别说明，下述实施例中使用的试剂为常规市购或商业途径获得的试剂，除非特别说明，下述实施例中使用的方法和设备为本领域常规使用的方法和设备。
实施例1：
本实施例提供一种强化人工湿地去除水体微囊藻及毒素的方法，是应用外源微囊藻毒素降解菌和人工湿地生物膜土著微生物的共同作用提高人工湿地对藻类及其毒素的去除效率；所述应用外源微囊藻毒素降解菌方法是从爆发微囊藻水华的水体中富集并提高微囊藻毒素的浓度， 在富集过程中定期检测降解菌基因mlrA，确认降解菌的培养时间，培养至微囊藻毒素降解菌液中微囊藻毒素降解菌的浓度为1×10 6～2×10 6细胞/m 2以上后将所述菌液应用于人工湿地。
所述人工湿地采用潜流人工湿地，湿地基质分为三层结构，自下而上依次为底层、中间层和表层，底层为沸石，中间层为碎砾石，表层为高有机质泥炭土，种植多年生水生草本植物。
所述方法的流程如附图1所示。包括以下步骤：
S1.构建人工湿地系统；所述系统的基质三层结构，自下而上依次为底层、中间层和表层，底层为沸石，中间层为碎砾石，表层为高有机质泥炭土，种植覆盖多年生水生草本植物；
S2.富营养化湖水挂膜处理：人工湿地系统运行前，通过富营养化湖水对湿地进行挂膜，以富集湿地中土著微生物，加速生物膜的形成，有利于外源降解菌的固定；
S3.高、低藻毒素负荷污水滴灌：
所述高藻毒素负荷污水滴灌方法是：
S31.从长期爆发微囊藻水华的水体筛选微囊藻毒素降解菌，以微囊藻毒素作为唯一碳氮源进行富集，并逐步提高微囊藻毒素的浓度；在富集过程中定期检测测降解菌基因mlrA，以确认降解菌的培养时间；
S32.将富集、驯化的微囊藻毒素降解菌液以1×10 6～2×10 6细胞/m 2以上的浓度均匀撒至人工湿地基质的表层上，并翻耕，空置后备用(种植植物)；
所述低藻毒素负荷污水滴灌方法是
S33.将含微囊藻毒素的污水输入人工湿地系统，停留一段时间，与湿地基质充分接触后作为出水排出人工湿地系统；
S4.定时取出水样，分析其中营养盐浓度、微囊藻细胞数及毒素含量；如果未达标，将出水停留处理后返回步骤S3；达标则进行排放。
步骤S4驯化之后的微囊藻毒素降解菌液用LB培养基(组成：蛋白胨5.0g，酵母浸出液2.5g，NaCl 5.0g，灭菌超纯水500mL，调节pH为7.0)培养，并用生理盐水重新悬浮后用于投加喷撒。
其中，菌落测序：采集长期爆发微囊藻水华湖泊的水体表层水样，用
Figure PCTCN2018080411-appb-000001
DNA Isolation Kit(MO-BIO,USA)提取试剂盒提取水样DNA，然后进行PCR扩增。
扩增的条件为：(1)反应体系(25μL)包括DNA模版2.0μL，10×PCR buffer 2.5μL，MgCl 2(25μM)2.0μL，dNTPs(2.5mM)2.0μL，PCR正、反向引物(10μmol/L)各0.5μL，Tap DNA聚合酶(5U/μL)0.2μL，并用灭菌去离子水补足。所用引物信息如表1。
(2)反应体系通过96-well C100TM thermal cycler PCR仪(Bio-Rad，USA)进行PCR扩增。PCR反应条件：(1)95℃3min；(2)95℃30s，62℃1min，72℃1min，35cycles；(3)72℃7min，4℃forever。
表1 mlrA基因PCR和qPCR反应引物序列
Figure PCTCN2018080411-appb-000002
扩增后通过含1×GelRed核酸染料的1.5％琼脂糖凝胶进行电泳，如附图3所示。并凝胶成像仪(Bio-Rad，USA)观察，待观察到微囊藻毒素降解菌mlrA预期条带，采用AxyPrep DNA凝胶回收试剂盒(AXYGEN)对目标条带进行切胶回收和纯化，纯化产物经PCR进一步检验其准确性，同时将纯化后产物测序。
菌液富集与培养操作为：确认菌源中存在微囊藻毒素降解菌后以实验室提取的粗微囊藻毒素作为唯一碳氮源进行富集，并逐步提高微囊藻毒素的浓度，在富集过程中定期检测测降解菌基因mlrA，以确认降解菌可以培养。具体步骤为：首先，取少量水华湖泊水接种到LB培养液中培养1～2d，然后从富集菌液中取1.0mL接种到含实验室提取的微囊藻毒素的无机盐培养基(组成：CuCl 2，FeSO 4、ZnCl 2、MnCl 2各0.0005g，CaCl 2 0.02g，MgSO 4·7H 20 1.0g，NaCl 1.0g，KH 2PO 4 0.5g，K 2HPO 4 4.0g，灭菌超纯水1000mL，调节pH为7.5)中，恒温振荡(120r/min，30℃)。每隔3天按10％的接种量接种到新的无机盐培养基中，并逐渐提高选择性培养基中微囊藻毒素的浓度(1～20mg/L)，使微囊藻毒素降解菌逐渐富集，待最后一次富集后，离心收集菌体，并用缓冲液清洗，重新用生理盐水(9‰)悬浮菌体，同时通过实时荧光定量PCR方法检测mlrA基因拷贝数之后再投入湿地。
测序比对：对筛选的菌落的mlrA进行测序，并与BLAST对比，发现该降解菌基因片段与已报道鞘氨醇单胞菌USTB-05具有的mlrA基因相似度高达99％，进一步确认筛选出微囊藻毒素降解菌，测序结果如表SEQ ID NO：1所示。
本实施例同时提供一种优选的人工湿地系统，如附图2所示。包括潜流人工湿地、微囊藻毒素降解菌液输送装置2和含微囊藻毒素污水的输送装置3，所述潜流人工湿地包括湿地主体、进水装置11和排水装置15，所述湿地主体内装填湿地基质，进水装置11设置 于湿地主体侧壁，优选设置于侧壁的顶部。排水装置15设置于湿地主体的底部；所述进水装置分别连接含微囊藻毒素污水的输送装置3和微囊藻毒素降解菌液的输送装置2。
所述系统还包括出水取样装置和污水停留处理装置，所述出水取样装置连接排水装置，所述污水停留处理装置一端连接排水装置，另一端连接湿地主体(图2中未标示)。
所述进水装置11采用滴灌方式。
所述湿地基质分为三层结构，自下而上依次为表层121、中间层122和底层123，底层123为沸石，中间层122为碎砾石，表层121为高有机质泥炭土，种植多年生水生草本植物14。
所述底层123厚度为5～9cm；所述沸石的粒径为1～2cm。
所述中间层122的厚度为15～20cm；所述碎砾石的粒径为0.5～1cm。
所述表层的厚度为5～10cm。表层采用高有机质泥炭土有利于微生物固定和繁殖。
所述多年生水生草本植物为黄菖蒲等。
所述湿地主体可以采用圆柱形结构。
实施例2本发明对氮磷等营养盐的去除效果试验
构建和利用实施例1所述的潜流人工湿地系统及方法，利用10L的PVC塑料桶构建有效容积为2.5L的人工湿地，填充基质从上往下依次为5cm土壤层，15cm砾石(粒径5mm)层，砾石在填充之前用自来水冲洗干净，底层为5cm大颗粒沸石(20mm)填充，以防出水堵塞,每个装置中均匀种植3株黄菖蒲。
装置运行：装置建成后用富营养化湖泊水体进行灌溉培养1年，人为添加营养盐，进水中氮磷等营养盐浓度见表2所示：
表2进水水质
Figure PCTCN2018080411-rpde-000003
氮磷去除效果：构建的人工湿地系统对氮磷营养盐去除效果稳定。系统TN的平均去除率为67.5％～75.3％，NH 4 +-N的平均去除率为52.6％～66.3％，NO 3 --N的平均去除率为91.6％～94.2％，TP的平均去除率为76.0％～92.8％。
实施例3：强化人工湿地对藻细胞和溶解性微囊藻毒素LR的去除试验
构建和利用实施例1所述的潜流人工湿地系统及方法，将降解菌复合菌液均匀倒置在湿地表 面土壤，采用翻耕的方式使复合菌液与土壤基质混合均匀，并驯化一个星期。初始微囊藻毒素MC-LR浓度分别设置为3.61μg/L、6.80μg/L、16.07μg/L。一次性进水后，在湿地中停留7d，分别于0h，6h，12h，24h，48h，72h，96h，120h，144h，168h采集1mL水样用于测定EMC-LR.
湿地系统的最佳水力停留时间为3d，在此水力停留时间下，人工湿地系统对产毒藻细胞(胞内毒素IMC-LR)的去除率为73.5％～95.4％，对胞外微囊藻毒素(EMC-LR)的去除率为87.5％～98.4％，可有效降低MC-LR含量至饮用水安全标准1.0μg/L以下；进一步延长湿地系统水力停留时间，可增强MC-LR的去除效果至低于检出限(0.10μg/L)。向湿地中投加微囊藻毒素复合降解菌能显著提高湿地系统对EMC-LR的去除速率(P<0.05)，缩短半衰期至22h，表明生物强化人工湿地能够更有效应急处理微囊藻毒素污染
为克服现有水体微囊藻及毒素处理方法的不足，本发明提供利用外源微囊藻毒素降解菌强化人工湿地去除水体微囊藻及毒素的方法，通过湿地中土著微生物和外源投加微囊藻毒素降解菌的生物降解作用加强湿地系统对微囊藻毒素的无害化处理，体现操作简便，经济快捷、高效灵活且无二次污染的优势，本发明实用性强，适应饮用水源和富营养化水体的处理需求，同时可以很好地与现有的水处理工艺对接。

Claims (10)

  1. 一种强化人工湿地去除水体微囊藻及毒素的方法，其特征在于，是应用外源微囊藻毒素降解菌和人工湿地生物膜土著微生物的共同作用提高人工湿地对藻类及其毒素的去除效率；所述应用外源微囊藻毒素降解菌方法是从爆发微囊藻水华的水体中富集并提高微囊藻毒素的浓度，在富集过程中定期检测降解菌基因mlrA，确认降解菌的培养时间，培养至微囊藻毒素降解菌液中微囊藻毒素降解菌的浓度为1×10 6～2×10 6细胞/m 2以上后将所述菌液应用于人工湿地。
  2. 根据权利要求1所述强化人工湿地去除水体微囊藻及毒素的方法，其特征在于，所述人工湿地采用潜流人工湿地，湿地基质分为三层结构，自下而上依次为底层、中间层和表层，底层为沸石，中间层为碎砾石，表层为高有机质泥炭土，种植多年生水生草本植物。
  3. 根据权利要求1所述强化人工湿地去除水体微囊藻及毒素的方法，其特征在于，所述应用外源微囊藻毒素降解菌方法将实验室富集、驯化的微囊藻毒素降解菌以1×10 6～2×10 6细胞/m 2以上的浓度均匀撒至人工湿地的表层，并翻耕，空置后使用。
  4. 根据权利要求1所述强化人工湿地去除水体微囊藻及毒素的方法，其特征在于，所述方法还包括将含微囊藻毒素的污水输入人工湿地。
  5. 根据权利要求1所述强化人工湿地去除水体微囊藻及毒素的方法，其特征在于，所述方法包括以下步骤：
    S1.构建人工湿地系统；所述系统的基质三层结构，自下而上依次为底层、中间层和表层，底层为沸石，中间层为碎砾石，表层为高有机质泥炭土，种植覆盖多年生水生草本植物；
    S2.富营养化湖水挂膜处理：人工湿地系统运行前，通过富营养化湖水对湿地进行挂膜，以富集湿地中土著微生物，加速生物膜的形成，有利于外源降解菌的固定；
    S3.高、低藻毒素负荷污水滴灌：
    S31.从长期爆发微囊藻水华的水体筛选微囊藻毒素降解菌，以微囊藻毒素作为唯一碳氮源进行富集，并逐步提高微囊藻毒素的浓度；在富集过程中定期检测测降解菌基因mlrA，以确认降解菌的培养时间；
    S32.将富集、驯化的微囊藻毒素降解菌液以1×10 6～2×10 6细胞/m 2以上的浓度均匀撒至人工湿地基质的表层上，并翻耕，空置后备用；
    S33.将含微囊藻毒素的污水输入人工湿地系统，停留一段时间，与湿地基质充分接触后作为出水排出人工湿地系统；
    S4.定时取出水样，分析其中营养盐浓度、微囊藻细胞数及毒素含量；如果未达标，将出水停留处理后返回步骤S3；达标则进行排放。
  6. 一种实现权利要求1至5任一项所述强化人工湿地去除水体微囊藻及毒素的方法的系统，其特征在于，包括潜流人工湿地、微囊藻毒素降解菌液输送装置和含微囊藻毒素污水的输送装置，所述潜流人工湿地包括湿地主体、进水装置和排水装置，所述湿地主体内装填湿地基质，进水装置设置于湿地主体侧壁，排水装置设置于湿地主体的底部；所述进水装置分别连接含微囊藻毒素污水的输送装置和微囊藻毒素降解菌液的输送装置。
  7. 根据权利要求6所述的系统，其特征在于，所述系统还包括出水取样装置和污水停留处理装置，所述出水取样装置连接排水装置，所述污水停留处理装置一端连接排水装置，另一端连接湿地主体。
  8. 根据权利要求6所述的系统，其特征在于，所述进水装置采用滴灌方式。
  9. 根据权利要求6所述的系统，其特征在于，所述湿地基质分为三层结构，自下而上依次为底层、中间层和表层，底层为沸石，中间层为碎砾石，表层为高有机质泥炭土，种植多年生水生草本植物。
  10. 根据权利要求6所述的系统，其特征在于，所述底层厚度为5～9cm；；所述中间层的厚度为15～20cm；所述表层的厚度为5～10cm。
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