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CN111471609A - Pseudomonas with algae-lysing activity and application thereof - Google Patents

Pseudomonas with algae-lysing activity and application thereof Download PDF

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CN111471609A
CN111471609A CN201911373674.5A CN201911373674A CN111471609A CN 111471609 A CN111471609 A CN 111471609A CN 201911373674 A CN201911373674 A CN 201911373674A CN 111471609 A CN111471609 A CN 111471609A
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甘南琴
宋立荣
陈莉婷
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Abstract

The invention discloses pseudomonas (a) with algae-lysing activityPseudomonassp.) THW7 with the preservation number of CCTCC NO: M20191065, has higher algae-lysing efficiency, is prepared into a bacterial embedding pellet and then is put into a cyanobacterial bloom water sample, and is supplemented with glucose with a certain concentration as energy supplement, thereby having good effect of controlling the cyanobacterial bloom.

Description

具有溶藻活性的假单胞菌及其应用Pseudomonas with algicidal activity and its application

技术领域technical field

本发明属于微生物控藻领域,具体涉及一种具有溶藻活性的假单胞菌及其在控制蓝藻水华中的应用。The invention belongs to the field of microbial algae control, in particular to a pseudomonas with algaelytic activity and its application in controlling cyanobacterial blooms.

背景技术Background technique

近二十年来,我国淡水水体蓝藻水华整体呈现高生物量、大范围、高频次、高危害的特征。形成水华的蓝藻种类主要有微囊藻(Microcystis)、长孢藻(Dolichospermum)、束丝藻(Aphanizomenon)、浮丝藻(Planktothrix)、节球藻(Nodularia)、颤藻(Oscillatoria)等,微囊藻水华是我国最主要的水华类型,长孢藻水华和束丝藻水华次之。In the past two decades, cyanobacterial blooms in freshwater waters in my country have shown the characteristics of high biomass, large scale, high frequency and high harm. The cyanobacteria species that form blooms mainly include Microcystis, Dolichospermum, Aphanizomenon, Planktothrix, Nodularia, Oscillatoria, etc. Microcystis blooms are the most important type of blooms in China, followed by algal blooms and algal blooms.

蓝藻水华的肆意暴发直接或间接地影响人类健康,并引起水体生态系统结构和功能的失衡,俨然已成为我国当前乃至今后长时期内面临和亟待解决的重大水环境问题。研究和发展控藻抑藻关键技术,促进湖泊向健康稳定方向发展具有重要的理论和现实意义。与常规物理法、化学法控藻技术相比,基于现代生态环保理念的微生物控藻技术具有具有经济、特异、安全、维持水体生态平衡的优势,是一种利用细菌、放线菌、真菌、原生动物及病毒等生物控制藻华以解决水环境问题的生态修复技术。合理利用微生物法控制水华蓝藻,对改善水质、缓解水华危害有着十分重要的意义。The wanton outbreak of cyanobacterial blooms directly or indirectly affects human health and causes imbalances in the structure and function of water ecosystems. It is of great theoretical and practical significance to research and develop key technologies for algae control and algae suppression, and to promote the development of lakes in a healthy and stable direction. Compared with conventional physical and chemical algae control technologies, the microbial algae control technology based on modern ecological and environmental protection concepts has the advantages of economy, specificity, safety, and maintaining the ecological balance of water bodies. Ecological restoration technology for protozoa and viruses to control algal blooms to solve water environment problems. The rational use of microbial methods to control blooms and cyanobacteria is of great significance for improving water quality and alleviating the harm of algal blooms.

纵观国内外的研究,微生物控藻法中报道最多的是利用溶藻细菌控制蓝藻水华。但鉴于其实际溶藻效果易受水体生态系统中各种生物因子、非生物因子影响,且有些微生物的生态安全性有待进一步的科学论证,目前对于溶藻细菌的研究主要集中于分离鉴定、溶藻现象的描述及其溶藻方式、机理方面,对其野外应用方法的研究尚处于初级阶段,少有关于其成功应用的案例。因此,筛选出具有高效溶藻效能的溶藻细菌并初步尝试其实际应用的方法,对水体水华的治理有着十分重要的意义。Throughout the research at home and abroad, the most reported method of microbial algae control is the use of algicidal bacteria to control cyanobacterial blooms. However, in view of the fact that its actual algae-dissolving effect is easily affected by various biological and abiotic factors in the water ecosystem, and the ecological safety of some microorganisms needs further scientific demonstration, the current research on algicidal bacteria mainly focuses on isolation, identification, dissolution In terms of the description of algae phenomenon and the way and mechanism of algal dissolution, the research on its field application method is still in its infancy, and there are few cases of its successful application. Therefore, it is of great significance to screen out the algae-lytic bacteria with high algae-dissolving efficiency and initially try their practical application methods for the management of water blooms.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种具有溶藻活性的假单胞菌,该菌株具有较高的溶藻效率,将其制成菌体包埋小球用于控制蓝藻水华也具有较好的效果,使用寿命较长,可反复使用,安全环保。The purpose of the present invention is to provide a kind of Pseudomonas with algae-lytic activity, the strain has high algae-lysis efficiency, and it also has good effect when it is made into thalline-embedded pellets for controlling cyanobacterial blooms , Long service life, can be used repeatedly, safe and environmentally friendly.

为了实现上述目的,本发明采用以下技术方案:In order to achieve the above object, the present invention adopts the following technical solutions:

发明人从发生蓝藻水华的太湖采集水样,经分离纯化培养及溶藻效率筛选,得到一株溶藻细菌THW7,为G-菌,短杆状或略弯,长约1.0-8.0μm,宽约0.3-1.0μm。在LB固体平板上形成的菌落呈中央略微凸起的圆球状,乳白色,质地较疏松,易被接种环挑起,表面光滑、不透明、较湿润,边缘整齐。通过16S rDNA测序和形态学鉴定,该菌株属于假单胞菌属(Pseudomonas),命名为假单胞菌(Pseudomonas sp.)THW7,已于2019年12月18日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 20191065。The inventor collected water samples from Taihu Lake where cyanobacteria blooms occurred, and obtained a strain of algae-lytic bacteria THW7, which was a G-bacteria, short rod-shaped or slightly curved, about 1.0-8.0 μm in length, after separation, purification, culture and algal-lysis efficiency screening. The width is about 0.3-1.0 μm. The colonies formed on the LB solid plate are spherical with a slightly raised center, milky white, loose in texture, easy to be provoked by the inoculation ring, smooth, opaque, moist surface, and neat edges. Through 16S rDNA sequencing and morphological identification, the strain belongs to the genus Pseudomonas, named Pseudomonas sp. THW7, which has been deposited in the China Center for Type Culture Collection on December 18, 2019 , the deposit number is CCTCC NO: M 20191065.

假单胞菌(Pseudomonas sp.)THW7在控制蓝藻水华中的应用,其方法如下:The application of Pseudomonas sp. THW7 in the control of cyanobacterial blooms is as follows:

(1)收集假单胞菌菌体,洗涤后用无菌水重悬;(1) Collect Pseudomonas thalline, wash and resuspend with sterile water;

(2)将假单胞菌重悬液加入3%-4%的海藻酸钠溶液中,搅拌混匀后将混合液逐滴加入3.5%-4.5%的氯化钙溶液,交联反应后获得包埋小球,表面光滑,直径4-6mm;(2) Add the Pseudomonas resuspended solution to 3%-4% sodium alginate solution, stir and mix well, add 3.5%-4.5% calcium chloride solution dropwise to the mixed solution, and obtain after cross-linking reaction Embedding beads, smooth surface, diameter 4-6mm;

(3)将假单胞菌菌体包埋小球后投入蓝藻水华水体中,并在水体中添加1.0-1.2g/L葡萄糖。(3) Put the Pseudomonas cells into the cyanobacterial bloom water after embedding the pellets, and add 1.0-1.2 g/L glucose to the water.

与现有技术相比,本发明具有以下优点及有益效果:Compared with the prior art, the present invention has the following advantages and beneficial effects:

针对利用溶藻细菌实际控藻中培养耗费大且实际应用时对水体生态系统造成的额外营养负荷问题,本发明利用廉价易获得的海藻酸钠和氯化钙交联将菌体包埋,再辅加一定浓度的葡萄糖作为能源补充,投入藻液中进行溶藻实验,不仅对单细胞的铜绿微囊藻具有非常明显的溶藻效果,而且也能够使蓝藻水华显著下沉,是一种有效治理蓝藻水华的方法,提供了治理蓝藻水华的新思路。这种包埋小球收放自如,使用寿命较长,可反复使用,一方面简化了控藻操作流程,降低菌种培养耗费等成本,另一方面减少溶藻过程本身给水体带来的额外营养负荷,相对环保。Aiming at the problem of extra nutrient load caused by the use of algae-lysing bacteria to actually control algae and the additional nutrient load caused to the water ecosystem during practical application, the invention uses cheap and easily available sodium alginate and calcium chloride to cross-link to embed the bacteria, and then Adding a certain concentration of glucose as an energy supplement and putting it into the algae solution for algae-lysis experiments not only has a very obvious algae-lysis effect on the single-cell Microcystis aeruginosa, but also can make the cyanobacteria bloom significantly sink. The method of effective management of cyanobacterial blooms provides a new idea for the management of cyanobacterial blooms. The embedded pellets can be freely retracted, have a long service life, and can be used repeatedly. On the one hand, it simplifies the algae control operation process and reduces the cost of bacterial culture, and on the other hand, it reduces the extra cost caused by the algae dissolution process itself to the water body. Nutrient load, relatively environmentally friendly.

附图说明Description of drawings

图1为溶藻细菌的分离纯化流程图。Figure 1 is a flow chart of the separation and purification of algicidal bacteria.

图2为菌株THW7在LB固体平板上的菌落照片。Figure 2 is a photo of the colony of strain THW7 on LB solid plate.

图3为菌株THW7的透射电镜照片。Figure 3 is a transmission electron microscope photograph of strain THW7.

图4为菌株THW7的16S rDNA序列通过BLAST比对,相关序列于Bioedit 7.0.9.1进行序列比对分析,最后用MEGA6.0、采用邻接法(Neighbor-joining,重复度F1000)构建的系统发育树。Figure 4 shows the 16S rDNA sequence of strain THW7 was compared by BLAST, the related sequences were compared and analyzed in Bioedit 7.0.9.1, and finally the phylogenetic tree constructed by MEGA6.0 and Neighbor-joining method (repetition degree F1000) .

图5为菌株THW7的生长曲线。Figure 5 is the growth curve of strain THW7.

图6为THW7菌体的包埋小球照片。Figure 6 is a photograph of the embedded pellet of THW7 cells.

图7为单细胞铜绿微囊藻中投加THW7菌体的包埋小球后,第10d三角瓶中藻液的照片。Figure 7 is a photo of the algal liquid in the Erlenmeyer flask on the 10th day after adding the THW7 cell-embedded pellets to the unicellular Microcystis aeruginosa.

图8为THW7菌体的包埋小球对单细胞铜绿微囊藻的溶藻效率柱状统计图。Figure 8 is a histogram of the algicidal efficiency of THW7 cells embedded pellets on unicellular Microcystis aeruginosa.

图9为野外群体藻样中投加THW7菌体的包埋小球后,藻液叶绿素a浓度变化的柱状图。Fig. 9 is a bar graph showing the change of chlorophyll a concentration in algal fluid after adding THW7 cell-embedded pellets to algal samples in the field.

图10为野外群体藻样中投加THW7菌体的包埋小球后,三角瓶中藻液在7d时的照片。Figure 10 is a photograph of the algal liquid in the Erlenmeyer flask at 7d after adding the THW7 cell-embedded pellets to the field colony algae samples.

图11为野外群体藻样中投加THW7菌体的包埋小球后,三角瓶中藻液在20d时的照片。Figure 11 is a photo of the algal liquid in the Erlenmeyer flask at 20 d after adding the embedded pellets of THW7 cells to the algal samples in the field.

具体实施方式Detailed ways

以下结合附图对本发明的具体实施方法进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。本发明所述技术方案,如未特别说明,均为本领域的常规方案。所述试剂或材料,如未特别说明,均来源于商业渠道。The specific implementation method of the present invention will be described below with reference to the accompanying drawings, and the examples are only used to explain the present invention, and are not used to limit the scope of the present invention. The technical solutions of the present invention, unless otherwise specified, are conventional solutions in the art. The reagents or materials, unless otherwise specified, were obtained from commercial sources.

实施例1:假单胞菌(Pseudomonas sp.)THW7的分离纯化及培养Example 1: Isolation, purification and culture of Pseudomonas sp. THW7

1.假单胞菌THW7的分离纯化与保藏1. Isolation, purification and preservation of Pseudomonas THW7

溶藻细菌的分离纯化流程如图1所示。从太湖采集蓝藻水华水样,经0.8μm的滤膜(Millipore)过滤,上清用无菌水进行10倍梯度稀释(稀释级别视具体情况而定),取100μL各梯度的稀释液均匀涂布于LB固体平板,每个梯度三个平行,写上对应编号,于生化培养箱中30℃倒置培养48h。选取菌落分布较为均匀的平板,挑取不同形态的单菌落在LB固体平板分区划线分离纯化两次,得到纯培养的菌株,用甘油保藏法保存在-80℃冰箱。The isolation and purification process of algicidal bacteria is shown in Figure 1. The cyanobacteria bloom water samples were collected from Taihu Lake, filtered through a 0.8 μm filter membrane (Millipore), and the supernatant was diluted 10 times with sterile water (dilution level depends on the specific situation). Distributed on LB solid plate, three parallel gradients for each, write the corresponding number, and incubate in a biochemical incubator at 30°C upside down for 48h. Select a plate with a relatively even distribution of colonies, pick a single colony of different shapes, and separate and purify it twice on a LB solid plate to obtain a pure cultured strain, which is stored in a -80°C refrigerator by the glycerol preservation method.

挑取单克隆接种于LB固体斜面,待细菌菌落长出,用无菌水冲刷,吹打均匀制成菌苔洗脱液。以10%的体积比添加菌苔洗脱液到铜绿微囊藻(Microcystis aeruginosa PCC7806)藻液中,空白对照组藻液中加入等体积的无菌水,于24孔板中进行共培养。置于光照培养箱中,培养温度为25℃、光强为25μE、光暗周期比为12h∶12h。5d后观察,若藻液黄化则该菌为溶藻菌。Pick a single clone and inoculate it on the LB solid slant. When the bacterial colonies grow, rinse with sterile water, and pipette evenly to prepare the bacterial moss eluate. The bacterial moss eluate was added to the algal solution of Microcystis aeruginosa PCC7806 at a volume ratio of 10%, and an equal volume of sterile water was added to the algal solution of the blank control group for co-cultivation in a 24-well plate. The cells were placed in a lighted incubator, the culture temperature was 25°C, the light intensity was 25 μE, and the light-dark cycle ratio was 12h:12h. Observation after 5 days, if the algal liquid yellows, the bacteria are algicidal bacteria.

根据初筛结果,挑取溶藻菌的单克隆接种于LB液体培养基,于控温摇床中30℃、140rpm培养48h,以10%的体积比将细菌培养物添加到铜绿微囊藻(Microcystisaeruginosa PCC 7806)藻液中,以加入等体积的无菌水作为空白对照组,于100mL三角瓶中进行共培养。置于光照培养箱中,培养条件同上,6d后测定其叶绿素a的含量,计算溶藻效率。溶藻效率的计算公式如下:According to the preliminary screening results, single clones of alginolytic bacteria were picked and inoculated into LB liquid medium, cultured in a temperature-controlled shaker at 30 °C and 140 rpm for 48 h, and the bacterial culture was added to Microcystis aeruginosa at a volume ratio of 10% ( Microcystisaeruginosa PCC 7806) algae liquid, an equal volume of sterile water was added as a blank control group, and co-cultivation was carried out in a 100 mL conical flask. It was placed in a light incubator, and the culture conditions were the same as above. The formula for calculating the algae-dissolving efficiency is as follows:

溶藻效率(%)=[(C0-C)/C0]×100%Algae lysis efficiency (%)=[(C 0 -C)/C 0 ]×100%

公式中C0为处理组初始的叶绿素a浓度,C为处理组测定时的叶绿素a浓度。In the formula, C 0 is the initial chlorophyll a concentration of the treatment group, and C is the chlorophyll a concentration of the treatment group when measured.

通过以上方法,筛选出一株溶藻效率较高的菌株,该菌株在第6天的溶藻效率为84.61%,编号THW7。菌株THW7在LB固体平板上的菌落形态如图2所示。菌株THW7在LB固体平板上形成的菌落呈中央略微凸起的圆球状,乳白色,质地较疏松,易被接种环挑起,表面光滑、不透明、较湿润,边缘整齐。菌株THW7的透射电镜照片如图3所示,呈短杆状或略弯,长约1.0-8.0μm,宽约0.3-1.0μm。Through the above method, a strain with higher algae-dissolving efficiency was screened out. The algae-dissolving efficiency of this strain was 84.61% on the sixth day, and the number was THW7. The colony morphology of strain THW7 on LB solid plate is shown in Figure 2. The colony formed by strain THW7 on the LB solid plate was spherical with a slightly raised center, milky white, loose in texture, easy to be provoked by the inoculation ring, smooth, opaque, moist surface, and neat edge. The transmission electron microscope photo of strain THW7 is shown in Figure 3, which is short rod-shaped or slightly curved, about 1.0-8.0 μm long and 0.3-1.0 μm wide.

设计引物扩增其16S rRNA基因进行测序,然后进行BLAST比对,采用邻接法(Neighbor-joining,重复度F1000)构建系统发育树如图4所示,菌株THW7与蒂氏假单胞菌Pseudomonas teessidea type strain PR65T(AM419154.2)的亲缘关系最近,16S rDNA基因相似性为99.79%。从系统发育树可以看出,Pseudomonas属为独立分支,分子生物学鉴定菌株THW7属于假单胞菌(Pseudomonas)。Primers were designed to amplify the 16S rRNA gene for sequencing, followed by BLAST alignment, and a phylogenetic tree was constructed using the Neighbor-joining method (F1000 repetition), as shown in Figure 4. Strain THW7 and Pseudomonas teessidea The genetic relationship of type strain PR65T (AM419154.2) was the closest, and the 16S rDNA gene similarity was 99.79%. It can be seen from the phylogenetic tree that the genus Pseudomonas is an independent branch, and the strain THW7 is identified by molecular biology as belonging to Pseudomonas.

该菌株保藏于-80℃甘油中,命名为假单胞菌THW7,拉丁学名为Pseudomonassp.THW7(以下简称菌株THW7),已于2019年12月18日保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:M 20191065。The strain was stored in -80°C glycerol, named Pseudomonas THW7, Latin scientific name Pseudomonassp.THW7 (hereinafter referred to as strain THW7), has been deposited in the China Center for Type Culture Collection on December 18, 2019, preservation The number is CCTCCNO: M 20191065.

2.假单胞菌THW7的培养2. Culture of Pseudomonas THW7

挑取单克隆于新鲜无菌LB培养基(表1)中扩大培养。扩大培养后的菌液再接种到新鲜LB液体培养基中,控制其初始OD600nm为0.088,在30℃,140rpm条件下培养,定时取样,用分光光度计测定其OD600nm。绘制的生长曲线如图5所示,菌株THW7的生长分为三个阶段,分别为停滞期(0-1h),对数期(1-24h),稳定期(24-34h)。34h之后,菌株THW7进入衰亡期。Single clones were picked and expanded in fresh sterile LB medium (Table 1). The expanded bacterial liquid was then inoculated into fresh LB liquid medium, and its initial OD 600nm was controlled to be 0.088, cultured at 30°C, 140 rpm, and the samples were periodically sampled, and the OD 600nm was measured with a spectrophotometer. The drawn growth curve is shown in Figure 5. The growth of strain THW7 is divided into three stages, namely the stationary phase (0-1h), the logarithmic phase (1-24h), and the stationary phase (24-34h). After 34h, strain THW7 entered the dying stage.

表1 LB培养基成分Table 1 Composition of LB medium

Figure BDA0002338867140000051
Figure BDA0002338867140000051

实施例2:假单胞菌THW7在控制蓝藻水华中的应用Example 2: Application of Pseudomonas THW7 in the control of cyanobacterial blooms

取对数期(20h,OD600nm=1.8)的菌液室温离心(5000rpm,6min),收集菌体,用无菌水清洗2-3次以洗去菌体表面的LB培养基。所获菌体加入无菌水(体积尽可能小)重悬,制得菌体重悬液备用。Take the bacterial liquid in log phase (20h, OD600nm =1.8) by centrifugation at room temperature (5000rpm, 6min), collect the bacteria, and wash with sterile water for 2-3 times to wash off the LB medium on the surface of the bacteria. The obtained bacterial cells were resuspended by adding sterile water (the volume was as small as possible) to prepare a bacterial resuspended suspension for later use.

将菌体重悬液加入到灭菌后的质量浓度为3%的海藻酸钠溶液中,用洁净玻璃棒搅拌混匀后用注射器将该混合液逐滴打入质量浓度为4%的氯化钙溶液中,交联30min。无菌水清洗包埋小球3-5次洗去表面的氯化钙溶液,制得直径约为4-6mm的菌体包埋小球备用,如图6所示。Add the bacterial suspension to the sterilized sodium alginate solution with a mass concentration of 3%, stir and mix with a clean glass rod, and then inject the mixture dropwise into calcium chloride with a mass concentration of 4% with a syringe. solution, cross-linking for 30min. Sterile water is used to wash the embedded beads for 3-5 times to remove the calcium chloride solution on the surface, and the bacteria embedded beads with a diameter of about 4-6 mm are prepared for later use, as shown in FIG. 6 .

将对数生长期的铜绿微囊藻(Microcystis aeruginosa PCC 7806)藻液分装于250mL三角瓶中,每瓶150mL,共9瓶,3瓶为一组,分别为空白对照组、葡萄糖组、菌体包埋小球组。取浓度为30g/L的葡萄糖溶液加入到葡萄糖组和菌体包埋小球组的藻液中,使其终浓度为1g/L,将菌体包埋小球投至对应实验组的藻液中,置于光照培养箱中培养,培养条件同上。10d后测定其叶绿素a的含量,计算溶藻效率。第10d三角瓶中藻液的照片如图7所示,葡萄糖对藻无影响,葡萄糖组同空白对照组的藻生长状况良好,藻液浓度升高并且颜色保持绿色,而菌体包埋小球组藻生长受到严重抑制,藻液浓度降低,颜色泛白,藻细胞大量死亡。实验中所用的葡萄糖对藻生长无影响且满足了菌体生长所需能源的需求,同时,比起蛋白胨、酵母粉或牛肉膏等富营养的菌体培养液成分,葡萄糖相对寡营养,是一种相对简单且环境友好型添加物。The algal liquid of Microcystis aeruginosa PCC 7806 in the logarithmic growth phase was divided into 250mL conical flasks, each 150mL, a total of 9 bottles, 3 bottles as a group, respectively blank control group, glucose group, bacteria Body-embedded pellet group. Take the glucose solution with a concentration of 30g/L and add it to the algae liquid of the glucose group and the cell-embedded pellet group to make the final concentration of 1g/L, and then put the cell-embedded pellets into the algae liquid of the corresponding experimental group. , placed in a lighted incubator and cultured under the same conditions as above. After 10 days, the chlorophyll a content was measured, and the algae-dissolving efficiency was calculated. The photo of the algal liquid in the Erlenmeyer flask on the 10th day is shown in Figure 7. Glucose has no effect on the algae. The algae in the glucose group and the blank control group grow well. The concentration of the algal liquid increases and the color remains green. The growth of algae in the group was severely inhibited, the concentration of algal liquid was reduced, the color was white, and the algal cells died in large numbers. The glucose used in the experiment has no effect on the growth of algae and meets the energy requirements for bacterial growth. At the same time, compared with nutrient-rich bacterial culture liquid components such as peptone, yeast powder or beef extract, glucose is relatively oligonutrient and is a A relatively simple and environmentally friendly additive.

一轮溶藻实验结束后,收获菌体包埋小球,用无菌水清洗小球表面4-5次,留待下一轮溶藻实验,实验步骤同上。重复此过程至菌体包埋小球无溶藻活性或溶藻效率不佳时为止。分析菌体包埋小球每次溶藻效能的变化及使用寿命。一共做了六次溶藻实验,每次的溶藻效率柱状统计图如图8所示。前四次溶藻效果呈现一个略微上升的趋势,但无显著性差异,溶藻效率在45%-65%之间,其中第四次溶藻效率为62.85%。第五次、第六次溶藻效率和第四次相比均有不同程度的下降,但仅第六次时的降低具有显著性,溶藻效率降至37.32%。由此看来,菌体被包埋成这种小球后,可反复收获用于溶藻,利用1-5次效果最佳,第6次开始溶藻效率显著性降低,以第一次溶藻实验开始至第五次实验结束的这段时间作为包埋小球的使用寿命,为68天。After one round of algae-lysis experiment, harvest the thalli-embedded pellets, wash the surface of the pellets with sterile water 4-5 times, and leave it for the next round of algae-lysis experiment. The experimental steps are the same as above. Repeat this process until the microbial cell-embedded beads have no algicidal activity or the algicidal efficiency is poor. The changes of the algae-dissolving efficiency and the service life of the bacteria-embedded beads were analyzed. A total of six algae-dissolving experiments were performed, and the histogram of the algae-dissolving efficiency for each time is shown in Figure 8. The first four algae-dissolving effects showed a slight upward trend, but there was no significant difference. The algae-dissolving efficiency was between 45% and 65%, and the fourth algae-dissolving efficiency was 62.85%. Compared with the fourth time, the algae-dissolving efficiency of the fifth and sixth times all decreased to different degrees, but only the sixth time had a significant decrease, and the algae-dissolving efficiency dropped to 37.32%. From this point of view, after the bacterial cells are embedded into such pellets, they can be harvested repeatedly for algae lysis. The best effect is to use 1-5 times, and the algae lysis efficiency will decrease significantly from the 6th time. The period from the beginning of the algae experiment to the end of the fifth experiment was taken as the service life of the embedded pellet, which was 68 days.

采集野外蓝藻水华水样,分装至洁净无菌的1L三角瓶中,每瓶装600mL,共6瓶,3瓶为一组,分别为对照组、处理组。实验初始,向处理组投加菌体包埋量为40mL的THW7菌体包埋小球,7d后观察实验现象,并取样测定叶绿素a浓度。与此同时,补加一定体积的葡萄糖溶液,使其终浓度为1g/L,继续观察实验过程中藻的变化情况,20d时取样测定叶绿素a浓度,分析溶藻效果。图9为向野外群体藻样中投加THW7菌体的包埋小球后,藻液叶绿素a浓度变化的柱状图,图10、图11为向野外群体藻样中投加THW7菌体的包埋小球后,三角瓶中藻液在不同时间点(7d、20d)的照片。实验发现在不添加任何能源的条件下,菌体包埋小球对野外群体藻无溶藻效果,但培养过程中可见部分藻下沉至底部的现象,还有一部分藻贴在三角瓶瓶壁。补给葡萄糖后,处理组藻群体几乎全部沉至底部,漂浮在水表面的群体极少。水体叶绿素a含量开始降低,同第7d相比,20d时处理组叶绿素a浓度降低了31.50%。这表明,该菌体包埋小球在野外水样藻华控制中具有较好的效果,实用性较强,尤其是在使大量群体藻下沉方面效果显著。同时,该菌体包埋小球可收放自如,使用寿命较长,可反复使用,一方面简化了控藻操作流程,降低菌种培养耗费等成本,另一方面减少溶藻过程本身给水体带来的额外营养负荷,相对环保。Collect water samples of wild cyanobacteria blooms and pack them into clean and sterile 1L conical flasks, each containing 600mL, a total of 6 bottles, and 3 bottles form a group, which are respectively the control group and the treatment group. At the beginning of the experiment, 40 mL of THW7 bacteria-embedded pellets were added to the treatment group. After 7 days, the experimental phenomenon was observed, and samples were taken to determine the concentration of chlorophyll a. At the same time, a certain volume of glucose solution was added to make the final concentration 1g/L, and the changes of algae during the experiment were continued to be observed. At 20 d, samples were taken to measure the concentration of chlorophyll a to analyze the algae-dissolving effect. Fig. 9 is a bar graph showing the change of chlorophyll a concentration in algal liquid after adding the embedded pellets of THW7 cells to the algae samples in the field group. Photos of the algal fluid in the triangular flask at different time points (7d, 20d) after burying the pellets. The experiment found that without adding any energy, the bacteria-encapsulated pellets had no algae-dissolving effect on the algae in the field, but some algae could be seen sinking to the bottom during the cultivation process, and some algae were attached to the wall of the triangular flask. . After supplying glucose, almost all algal colonies in the treatment group sank to the bottom, and very few colonies floated on the water surface. The content of chlorophyll a in the water began to decrease. Compared with the 7th day, the chlorophyll a concentration of the treatment group decreased by 31.50% on the 20th day. This shows that the bacteria-embedded pellets have a good effect in the control of algal blooms in field water samples, and have strong practicability, especially in sinking a large number of groups of algae. At the same time, the bacteria-embedded pellet can be retracted freely, has a long service life, and can be used repeatedly. On the one hand, it simplifies the algae control operation process, reduces the cost of bacterial culture and other costs, and on the other hand reduces the algae dissolution process itself. The additional nutrient load brought by it is relatively environmentally friendly.

以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (5)

1. Pseudomonas (A) with algicidal activityPseudomonassp.), characterized in that the preservation number of the pseudomonas is CCTCC NO: M20191065.
2. An embedded pellet comprising the pseudomonad of claim 1.
3. The method for preparing the pseudomonas embedded pellet as claimed in claim 2, which comprises the following steps:
(1) collecting pseudomonas thallus, washing and re-suspending with sterile water;
(2) adding the pseudomonas heavy suspension into a 3-4% sodium alginate solution, uniformly stirring, dropwise adding the mixed solution into a 3.5-4.5% calcium chloride solution, and performing crosslinking reaction to obtain an embedded pellet with a smooth surface and a diameter of 4-6 mm.
4. The use of the pseudomonads of claim 1 for the control of cyanobacterial bloom.
5. The use as claimed in claim 4, wherein the Pseudomonas is prepared into bacteria embedding pellet, and then is thrown into cyanobacterial bloom water body, and 1.0-1.2 g/L glucose is added into the water body.
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