CN111165492A - 乙酰丙酮在抑制蓝藻生长中的应用 - Google Patents
乙酰丙酮在抑制蓝藻生长中的应用 Download PDFInfo
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- CN111165492A CN111165492A CN202010025079.9A CN202010025079A CN111165492A CN 111165492 A CN111165492 A CN 111165492A CN 202010025079 A CN202010025079 A CN 202010025079A CN 111165492 A CN111165492 A CN 111165492A
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Images
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
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- Y02W10/37—Wastewater or sewage treatment systems using renewable energies using solar energy
Abstract
本发明提供了乙酰丙酮在抑制蓝藻生长中的应用,即将在蓝藻生长水域投加乙酰丙酮,投加量不低于7mg/L,以控制藻的生长;乙酰丙酮通过作用于光合系统控制藻的生长,效果优于常见的抑藻剂(如焦性没食子酸、没食子酸和蒽醌等),并且对包括大肠杆菌、沙门氏菌、枯草芽孢杆菌、金黄色葡萄球菌等四种常见菌在内的细菌无害,具有环境友好性,易于推广应用。
Description
技术领域
本发明涉及抑藻剂领域,特别是乙酰丙酮在抑制富营养化水体中蓝藻生长的应用。
背景技术
近年来水体富营养化导致的蓝藻水华爆发严重影响了人们的生活。蓝藻水华不仅会引起其他水生生物死亡,改变水生生态环境,还直接关系着人类的供水安全。蓝藻一般分为产毒品系(如FACHB-905)和非产毒品系(FACHB-469),其中产毒品系会释放对肝脏具有毒性的微囊藻毒素。铜绿微囊藻是水华爆发时最为典型的藻类,也是最常见的一种有毒蓝藻,其生存能力强,难去除,因此,有效控制水体中铜绿微囊藻的生长具有重要的意义。
对于突发性的蓝藻水华,常用的方法为物理法或者化学法。物理法主要通过打捞等物理方式收集藻体,成本较高,在大水域的实施较为困难。化学法中最常用的是化学药剂法,即向水体中投加抑藻剂。目前研究较多的抑藻剂为化感物质,即利用其他生物体内产生的非营养性物质来控制藻的生长。如穗花狐尾藻分泌的酚酸类和脂肪酸类的物质以及轮叶黑藻分泌的N-苯基-2-萘胺都可以控制铜绿微囊藻的生长(“化感效应及其对藻类光合作用影响的研究进展”,环境科学与技术,2019,42(4),43-52)。部分化感物质虽然可以达到很好的抑杀蓝藻效果,但是由于自然水体中化感物质的有效成分不高,提纯化感物质极大地增加了控制水华的成本,导致化感抑藻剂的应用受到很大限制。
蒽醌、没食子酸、焦性没食子酸以及血根碱是目前常见的抑藻剂,这些抑藻剂均具有抗菌性(“焦性没食子酸修饰的多壁碳纳米管的抗菌性研究”,2013年学术年会论文摘要,天津生物医学工程;“没食子酸药理作用的研究进展”,中国医院药学杂质,2017,37,94-98;“蒽醌类化合物抗菌活性及其机制研究进展”,中国新药杂质,2016,25,2450-2455;“血根碱体外抑菌作用及其对细菌生物被膜的影响”,动物保健,2012,48,67-70),在抑制蓝藻生长的同时影响其他生物。
乙酰丙酮是一种小分子双酮,其标准命名为2,4-戊二酮,是一种重要的化工及医药中间体,主要应用于医药工业、兽药、饲料添加剂、催化剂、助催化剂的制备及无机材料的处理。专利CN 106519756 B公开了一种杀菌抗霉内墙纳米涂料其原料之一即为乙酰丙酮;专利CN 101454267 B公开了一种以香草醛和乙酰丙酮为原料缩合除水制备姜黄素的方法。专利CN 108217868 A公开了一种复合除藻剂的制备方法,其中乙酰丙酮作为水解抑制剂用于除藻剂胚体组分纳米二氧化钛的制备,该复合抑藻剂将化感物质及硫酸铜为抑藻物质固定到除藻剂胚体中,通过絮凝、光催化二氧化钛产生自由基、以及胚体中固定的抑藻物质的综合作用来控制藻的生长;根据乙酰丙酮的亨利定律常数(25℃时为2.35x10-6atm-cu m/mol)计算得到,其在模型河和模型湖的挥发半衰期分别为16天和120天(Handbook ofchemical property estimation methods.1990,pp.15-1至15-29)。通过实验确定乙酰丙酮在pH6.4时的水生氧化速率为9.9X10-9L mol-1s-1,基于此速率和连续日光照射下水中羟基自由基的浓度(1X10-17M),估计乙酰丙酮在水中氧化的半衰期为81天(Handbook ofchemical property estimation methods.1990,pp.7-4,7-5,8-12)。乙酰丙酮的生物富集因子(BCF)为3,说明其生物富集潜力很低(Exploring QSAR.Hydrophobic,Electronic,andSteric Constants.1995,p.14)。已有研究表明浓度高至50mg/L的乙酰丙酮及其光解产物对活性污泥具有生物友好性(“Fate and implication of acetylacetone inphotochemical processes for water treatment”,Water Res.2016,101,233-240);乙酰丙酮对水生动物大型蚤的半致死浓度高于50mg/L(“Feasibility of the UV/AA processas a pretreatment approach for bioremediation of dye-laden wastewater”,Chemosphere 2018,194,488-494)。上述数据表明乙酰丙酮应用于自然水体中不会造成不利的环境影响;虽然乙酰丙酮被广泛用作合成杀虫剂和杀菌剂的原料,但是迄今尚未见将其用作抑藻剂的报道。
发明内容
本发明针对目前缺乏经济有效的控制蓝藻水华的方法这一问题,提供了一种乙酰丙酮在抑制蓝藻生长中的应用,具体而言,上述发明目的是通过以下技术方案实现的:
首先,本发明提供了一种乙酰丙酮在抑制蓝藻生长中的应用;上述蓝藻包括铜绿微囊藻中产毒型菌株FACHB-905和不产毒型菌株FACHB-469。
其次,本发明提供了一种含有乙酰丙酮的蓝藻抑制剂。
第三,本发明还提供了一种抑制蓝藻生长的方法,其具体步骤如下:向蓝藻水域中投加乙酰丙酮,以抑制蓝藻生长;其中,乙酰丙酮的投加量不低于7mg/L,权利要求1中所述的铜绿微囊藻的有效控制范围为小于2.59x107cells/ml。
所述的铜绿微囊藻包括产毒的FACHB-905和不产毒的FACHB-469,密度适用范围为小于2.59x107cells/ml,其光密度与浓度之间存在如下关系:
Y=25.561X+0.3268;
其中Y为藻浓度(单位:106cells/ml),X为藻液的光密度值OD680。
本发明与常见抑藻剂(蒽醌、赖氨酸、2-甲基乙酰乙酸乙酯、没食子酸、焦性没食子酸和血根碱)做了比较,结果表明乙酰丙酮作用于铜绿微囊藻的效果优于其他抑藻剂。
本发明选取了常见的四种菌(大肠杆菌、沙门氏菌、枯草芽孢杆菌和金黄色葡萄球菌),发现6-1536mg/L的乙酰丙酮对上述菌种无抑制效果,具有环境友好性。
本发明提出的抑藻方法的作用机理为:乙酰丙酮通过抑制铜绿微囊藻的光合作用导致藻细胞死亡。
本发明与现有的物理和化学的抑杀蓝藻方法相比,更为经济有效。乙酰丙酮不仅有较好的抑藻效果,且在使用浓度范围内对活性污泥、大型蚤及一些常见细菌都具有生物友好性。将乙酰丙酮作为抑藻剂抑杀蓝藻是一种成本低廉、环境友好的控制藻生长的方法。
附图说明
图1为实施例AA抑制铜绿微囊藻(FACHB-905)生长过程中藻光密度变化情况示意图;
图2为实施例AA抑制铜绿微囊藻(FACHB-469)生长过程中藻光密度变化情况示意图;
图3为实施例不同初始浓度的铜绿微囊藻在15mg/L的AA作用下的藻光密度变化情况示意图;
图4为实施例AA与其他常见抑藻剂的效果对比情况示意图;
图5为实施例AA对不同类型的菌生长影响过程中菌光密度变化情况示意图;
图6为实施例AA抑制铜绿微囊藻生长过程中光合系统实际光化学量子产量变化情况示意图。
图7为自然光照条件下AA抑制铜绿微囊藻生长实验结果示意图。
具体实施方式
下面结合实施例对本发明进行详细的说明。
(1)实施例涉及试剂
实施例中所用的乙酰丙酮(以下简称AA,南京化学试剂)和焦性没食子酸(阿拉丁)为分析纯级,蒽醌(阿法埃莎)、L-赖氨酸(阿拉丁)和血根碱(麦克林)的纯度为98%,没食子酸(国药)的纯度为99%,2-甲基乙酰乙酸乙酯(阿拉丁)为化学纯级;
所用藻种为铜绿微囊藻高产毒株(FACHB-905)和不产毒株(FACHB-469),由中科院水生生物所提供,采用BG-11培养基培养,BG-11培养基的主要成分为:NaNO3,1.5g/L;K2HPO4·3H2O,0.04g/L;MgSO4·7H2O,0.075g/L;CaCl2·2H2O,0.036g/L;柠檬酸,0.006g/L;柠檬酸铁铵,0.006g/L;Na2EDTA,0.001g/L;NaCO3,0.02g/L;A5solution,0.1ml;其中A5solution的配方为:H3BO3,2.86g/L;MnCl2·4H2O,1.86g/L;ZnSO4·7H2O,0.22g/L;Na2MoO4·2H2O,0.39g/L;CuSO4·5H2O,0.08g/L;Co(NO3)2·6H2O,0.05g/L;
除特殊说明外,以下实施例中所用的藻种为FACHB-905高产毒株。所用菌种为大肠杆菌、沙门氏菌、枯草芽孢杆菌和金黄色葡萄球菌,均由南京大学生命科学学院提供,采用LB营养肉汤培养,LB营养肉汤的主要成分为:胰蛋白胨,10.0g/L;酵母浸粉,5.0g/L;氯化钠,10.0g/L。
(2)反应装置
铜绿微囊藻在光照培养箱(GZX-250BS-III)中培养,由上海新苗医疗器械制造有限公司提供,光照强度为3000LX,光暗时间比为12:12,温度设置为25℃。四种细菌均在生化培养箱中培养(SPX-150),由上海跃进医疗器械有限公司提供,温度设置为37℃。
(3)检测方法
铜绿微囊藻和其他菌的浓度使用紫外分光光度计(岛津,UV-2700)测量,分别以680nm和600nm处液体的光密度表征浓度,记作OD680和OD600。
叶绿素a的测量参照文献(“Potent removal of cyanobacteria withcontrolled release of toxic secondary metabolites by a titanium xerogelcoagulant”,Water Res.2018,128,341-349)中的方法,将收集到的藻液用0.7μm的GF/F玻璃滤膜(Whatman)过滤,收集藻细胞,在-4℃的冰箱中过夜冷冻,然后加入90%的丙酮过夜提取,提取液用高速冷冻离心机(天诺达,CT14RD)在10000rmp转速4℃下离心10min收集上清液,在630nm、663nm、645nm和750nm处检测吸光度,叶绿素a的含量计算公式如下:
其中A663、A750、A645及A750是溶液在663nm、750nm、645nm和750nm处的吸光度,Ve是提取溶液的体积,Vs是过滤溶液的体积,δ是比色皿的光程。抑藻剂对铜绿微囊藻的抑制率计算公式如下:
其中[chl-a]C是空白对照组(Control)中叶绿素a的含量,[chl-a]是加入抑藻剂的实验组中叶绿素a的含量。
实际光化学量子产量使用Water-PAM浮游植物分析仪(Walz)测量,取2.5ml藻液在光适应后测量Fm’和Fs,实际光化学量子产量计算公式如下:
φe=ΔF/Fm'=(Fm'-Fs)/Fm';
其中,φe是实际光化学量子产量,Fm`和Fs分别是藻液在光适应后最大叶绿素荧光和稳定叶绿素荧光(Effects of different algaecides on the photosyntheticcapacity,cell integrity and microcystin-LR release of Microcystis aeruginosa,Sci.Total Environ.2013,463,111-119)。
实施例1
(1)乙酰丙酮对高产毒株FACHB-905铜绿微囊藻的抑制效果
在无菌条件下向150ml的三角瓶中加入100ml的BG-11培养基,并接入藻种,使得OD680为0.2,并向三角瓶中投加不同体积的稀释100倍的AA原液,使得AA的投加量为5mg/L,7mg/L,10mg/L,15mg/L和20mg/L,同时设立不含AA的对照试验(Control);将藻液放入光照培养箱中培养,考虑到现有报道蓝藻最适宜的生长光照为2000-4000LX(参见“中国科学院淡水藻总库”,网址“http://algae.ihb.ac.cn/Products/ProductDetail.aspx?product=523”以及文献“光照对小球藻和铜绿微囊藻生长及叶绿素荧光的影响,西安文理学院学报,2019,22,73-77”),故本实施例中光照强度选择3000LX;在培养初期及培养0.5d,1d,1.5d,2d,3d,4d,5d后在无菌条件下取样检测OD680。
图1为5d内高产毒菌株的光密度变化情况,由图可见,AA在加入第1天对藻的生长没有显著影响,从第1.5天开始,AA浓度大于15mg/L的藻液中铜绿微囊藻的光密度开始下降,且继续培养的过程中藻持续死亡;AA浓度为20mg/L时,相较AA浓度为15mg/L的藻液中藻的生长曲线没有发生显著变化;10mg/L AA对藻的抑制作用弱于15mg/L AA,但是在培养时间范围内,还是持续表现出抑制藻生长的效果;AA浓度为7mg/L时,铜绿微囊藻的生长受到了抑制,但是藻依旧呈生长状态;5mg/L的AA对藻的生长相较Control组没有显著的影响。(2)乙酰丙酮对无毒株FACHB-469铜绿微囊藻的抑制效果
除藻种不同外,其他操作同步骤(1)。
图2为5天内无毒菌株的光密度变化情况,由图可知,与有毒菌株对AA反映相同的是,无毒菌株也在第1.5天受到了AA的影响,AA浓度越高,对藻生长的抑制作用越显著;当AA的投加量大于10mg/L时,藻的生长受到了显著的抑制,在后续培养过程中,藻持续死亡,当AA浓度升高至20mg/L时,藻生长曲线相较含有10mg/L和15mg/L AA的藻液没有明显变化;当AA浓度为7mg/L时,藻的生长抑制效果被显著减弱,但是与AA对产毒株效果不同的是,7mg/L的AA具有一定的抑藻作用;5mg/L的AA对无毒菌株仍然表现出生长抑制作用;由此可见,无毒菌株FACHB-469对于AA的耐受性较有毒菌株FACHB-905差,同浓度的AA对无毒菌株的作用更显著。
实施例2
在无菌条件下向150ml的三角瓶中加入100ml的BG-11培养基,并接入藻种,最终得到OD680为0.11,0.27,0.62和1.0的藻液,并向三角瓶中投加一定体积的稀释100倍的AA原液,使得AA的投加量为15mg/L,同时不同藻密度下都设立不含AA的对照试验(Control);将藻液放入光照培养箱中培养,在培养初期及培养1d,2d,3d,4d,5d后在无菌条件下取样检测OD680。
图3为不同初始浓度的藻在15mg/L AA浓度作用下5d内藻光密度变化情况,由图可知,藻的光密度值OD680从0.11变化到1.0时AA均对铜绿微囊藻表现出优异的抑制性能,说明AA对高密度的蓝藻(2.59x107cells/ml)作用效果依旧显著。
太湖是我国富营养化较严重的水体,2011-2017年太湖无锡水域藻密度的年均值均低于1.7x107cells/ml(太湖无锡水域水质变化特征及其原因分析,人民长江,2019,50,40-44),本实施例实验说明AA有望应用于实际水体中爆发的蓝藻水华。
实施例3
在无菌条件下向500ml的三角瓶中加入300ml的BG-11培养基,并接入藻种,使得OD680为0.2,并向三角瓶中投加AA、蒽醌、赖氨酸、2-甲基乙酰乙酸乙酯、没食子酸、焦性没食子酸以及血根碱的浓储备液,使得焦性没食子酸和血根碱的投加量分别为5和0.5mg/L,其他抑藻剂的投加量均为15mg/L,同时设立不含抑藻剂的对照试验(Control);将藻液放入光照培养箱中培养,在培养初期及培养1d,2d,3d后在无菌条件下取样检测叶绿素a的含量,以叶绿素a含量的变化来评估各种抑藻剂的效果。
图4为各种抑藻剂对铜绿微囊藻抑制率的对比情况,由图4可知,虽然部分抑藻剂在第2天就表现出较显著的抑藻效果,但是继续培养到第3天时AA对铜绿微囊藻的抑制率高于其他所有抑藻剂,实验中测试的抑藻剂中仅没食子酸的抑制率接近AA;需要指出的是,焦性没食子酸和血根碱由于引起水体色度和溶解度的因素而不宜于投放于环境水体中。
实施例4
在无菌条件下,取9个试管,并向其中加入4ml营养肉汤,编号1到9号,配制含有3072mg/L的AA肉汤储备液,取4ml加入9号试管,混匀9号试管后取4ml 9号试管中的溶液到8号试管中,以此类推,采用逐级稀释法稀释到2号试管,混匀后将2号试管中的溶液取4ml弃去,以1号试管为不加入AA的空白组(Control),最终各管中AA的浓度分别为0mg/L,6mg/L,12mg/L,24mg/L,96mg/L,192mg/L 384mg/L,768mg/L和1536mg/L;用营养肉汤稀释所用菌种的悬浮液至OD600为0.05,向9个试管中分别加入100μL稀释后的该菌悬浮液,用灭菌后的封口膜密封试管;完成上述操作后将菌液放入生化培养箱,温度设置为37℃,培养24h后测定各试管中的OD600。
图5为24h后经过不同浓度的AA处理后的大肠杆菌、沙门氏菌、枯草芽孢杆菌、金黄色葡萄球菌在600nm处光密度的变化,由图可见,AA浓度在6-1536mg/L范围内时四种菌都可以生长,且与不加AA的对照组相比,AA基本不影响四种菌的生长;大肠杆菌在12mg/L AA作用下生长速率较空白组稍慢,但当AA浓度提高至48mg/L时大肠杆菌的生长与空白组相比并没有显著差别;与空白组相比沙门氏菌在6-768mg/L AA的作用下生长速率加快,可能是AA作为碳源促进了沙门氏菌的生长,虽然AA浓度为1536mg/L时沙门氏菌的生长速率变慢,但是菌在24h内仍呈增长趋势;AA对枯草芽孢杆菌的作用与对大肠杆菌的类似;浓度高达768mg/L的AA对金黄色葡萄球菌仍有一定的促进生长作用。由以上四种菌对AA的耐受性可以看出AA在浓度小于1536mg/L的范围内都不会对这几种菌有杀害作用,且AA对部分菌有促进生长的作用。上述实施例说明AA对不含有光合系统的细菌没有毒害作用,进一步推测AA作为抑藻剂对于水中的浮游动物友好。
实施例5
在无菌条件下向150ml三角瓶中加入100ml的BG-11培养基,并接入藻种,使得OD680为0.2,并向三角瓶中加入不同体积稀释100倍的AA原液,使得AA的投加量分别为7mg/L,10mg/L,15mg/L,同时设立不含AA的对照试验(Control);将藻液放入光照培养箱中培养,在培养初期及培养0.5d,1d,1.5d,2d,3d,4d,5d后在无菌条件下取样进行叶绿素荧光参数的检测。
图6为AA抑制藻生长过程中藻的实际光化学量子产量变化,由图可知,在培养1天后铜绿微囊藻的实际光化学量子产量被AA显著抑制,且随着AA浓度的升高,这种抑制效果更加显著;继续培养的过程中,藻的实际光化学量子产量持续降低,生长到第2天时,浓度大于7mg/L的AA处理的藻液中藻的实际光化学量子产量为0;由实施例1可知在第1.5天时藻的生长才受到AA的抑制,而AA作用时藻的实际光化学量子产量在第1天时即受到抑制,说明AA作用于藻的光合系统,表现为AA导致藻的实际光化学量子产量显著降低,随后引起藻的死亡。
实施例6
在无菌条件下向150ml的三角瓶中加入100ml的BG-11培养基,并接入藻种,使得OD680为0.2,并向三角瓶中投加一定体积的稀释100倍的AA原液,使得AA的投加量为10mg/L和20mg/L,同时设立不含AA的对照试验(Control);将藻液置于朝阳的窗台处培养,在培养初期及培养0.5d,1d,1.5d,2d,3d,4d,5d后在无菌条件下取样检测OD680。
图7为5天内在太阳光下AA作用时藻光密度变化情况,由图7可知,在太阳光下对照组的藻液生长良好,而用10mg/L及20mg/L AA处理藻液的生长均显著受到了抑制,说明AA在太阳光下仍可以有效的抑制藻生长,可作为抑藻剂施入蓝藻生长水域。
Claims (5)
1.乙酰丙酮在抑制蓝藻生长中的应用。
2.根据权利要求1所述的应用,其特征在于,所述蓝藻包括铜绿微囊藻。
3.一种包含乙酰丙酮的抑藻剂。
4.一种抑制蓝藻生长的方法,其特征在于,具体步骤如下:向蓝藻生长水域投加乙酰丙酮,以抑制蓝藻生长。
5.如权利要求4所述的方法,其特征在于,所述乙酰丙酮的投加量不低于7 mg/L。
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