CN117343843A - 一株突变型莱茵衣藻、复合藻种及其土壤改良剂和应用 - Google Patents
一株突变型莱茵衣藻、复合藻种及其土壤改良剂和应用 Download PDFInfo
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- CN117343843A CN117343843A CN202310435680.9A CN202310435680A CN117343843A CN 117343843 A CN117343843 A CN 117343843A CN 202310435680 A CN202310435680 A CN 202310435680A CN 117343843 A CN117343843 A CN 117343843A
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- algae
- composite
- chlamydomonas reinhardtii
- composite algae
- soil conditioner
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- 239000011787 zinc oxide Substances 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
- C12N1/125—Unicellular algae isolates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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Abstract
本发明属于土壤重金属镉污染的修复技术领域,具体公开了一株突变型莱茵衣藻(Chlamydomonas reinhardtii),本发明还公开了一种复合藻种或其制得的复合藻种土壤改良剂。本发明还公开了复合藻种或其制得的复合藻种土壤改良剂的制备方法和在修复土壤重金属镉污染中的应用。本发明首次将获得的镉耐受型突变藻株扩大培养,并混合小球藻,利用细胞浓缩技术制备成复合藻种土壤改良剂,该复合藻种土壤改良剂施至镉污染的稻田中,原位治理,相较于其他植物修复、电化学吸附及换土等方法减少了二次污染及运输成本,而且获得了远远优于单株藻降低土壤重金属镉含量的技术效果,大幅高效降低稻米中的含镉量。
Description
技术领域
本发明属于土壤重金属镉污染的修复技术领域,具体涉及一株突变型莱茵衣藻、复合藻种及其土壤改良剂和应用。
背景技术
目前治理土壤重金属的方法主要有三种:物理修复、化学修复和生物修复。物理修复—利用土壤淋洗的方法对黏附在细微颗粒上的有毒物质进行水洗清除(USEPA,2007);利用1000℃高温或100℃低温进行原位加热和电磁波加热修复,该方法主要去除重金属汞,物理修复方法无复杂工艺流程,且无药剂添加,对环境不会造成二次污染,但由于其工艺需要用到喷淋或高温加热设备,因此对处理场地及成本的需求较高。化学修复—利用碳纳米管等材料吸附固体表面砷;利用胶体纳米金颗粒去除水体中的汞;利用硫及硫铁改性生物炭固定从而降低镉的生物利用度;氧化锌纳米颗粒(ZnO-NPs)单独或联合生物炭叶面喷施降低水稻根茎中镉含量,但是该类方法由于原材料的加入容易导致二次污染,且工程量大,处理土壤深度有限,造价昂贵。生物修复—筛选镉累积量相对较低的水稻品种以降低稻米中的镉含量,通过蜈蚣草、景天等植物修复减少土壤中的镉含量,微生物接种稻田以降低水稻中镉含量和促进水稻生长,但是由于我国镉污染土壤点位分布较为分散,根据各个地区环境去筛选品种需要多次进行试验,耗时较长,这些方法在实际应用中具有一定的局限性。而微藻作为生物土壤改良剂处理的方法具有造价低廉、吸附效率高、生物友好等特点,经前期试验对土壤施以富集后的微藻细胞可以吸附土壤中的重金属镉,且对土壤中种植的水稻结实无显著影响,为将来重金属镉污染土壤修复提供一种新的思路方法。
目前重金属镉污染修复方法各有利弊,成本投入、耗时及富集后的污染处理仍然需要后续跟踪研究,因此亟需一种成本低廉、见效快速的方法修复污染土壤,解决重金属污染的问题,利用微藻修复重金属污染的前景广阔。
发明内容
发明目的:本发明所要解决的技术问题是首先通过光照联合曝气培养装置获取莱茵衣藻细胞,然后利用1-甲基-3-硝基-1-亚硝基胍(MNNG)化学诱导衣藻细胞突变,并用筛选培养基选择性获得衣藻镉耐受型突变藻株。
本发明还要解决的技术问题是通过使镉耐受的突变藻株大规模繁殖,混合小球藻制成复合藻种土壤改良剂。
本发明最后要解决的技术问题是将该复合藻种土壤改良剂施至镉污染的稻田中,用于修复稻田重金属镉污染,以达到高效降低土壤重金属镉含量的目的。
技术方案:为解决上述技术问题,本发明提供了一株突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1,所述突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1于2023年03月30日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023436。
本发明内容还包括一种复合藻种或其制得的复合藻种土壤改良剂,所述复合藻种或其制得的复合藻种土壤改良剂包括所述的突变型莱茵衣藻(Chlamydomonasreinhardtii)MN1。
其中,所述复合藻种或其制得的复合藻种土壤改良剂还包括小球藻(Chlorellasp.)FACHB-10。
在所述复合藻种或其制得的复合藻种土壤改良剂中,所述突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1与小球藻(Chlorella sp.)FACHB-10的细胞数量的比值为1:1~2:1,两者细胞总浓度为108cells/mL。
本发明内容还包括所述的复合藻种的制备方法,包括以下步骤:
1)将突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1接入灭菌好的R培养基中进行无菌曝气培养,全光谱照明,光强3000~5000lux,光周期时长昼:夜=14:10,温度22~27℃,使其终浓度达到107cells/mL;
2)将小球藻(Chlorella sp.)FACHB-10接入灭菌好的BG11培养基中进行无菌培养,全光谱照明,光强3000~5000lux,光周期时长昼:夜=14:10,温度22~27℃,使其终浓度达到2×107cells/mL;
3)将步骤1)得到的突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1的悬液和和步骤2)得到的小球藻(Chlorella sp.)FACHB-10的悬液离心浓缩,以细胞数量1:1~2:1配比混合,并浓缩至两者细胞总浓度为108cells/mL,无菌罐装即制成复合藻种。
其中,步骤1)中所述R培养基的配制方法如下:硼酸0.3g,七水合硫酸锌0.3g,一水合硫酸锰0.091g,六水合氯化钴0.06g,二水合钼酸钠0.06g,五水合硫酸铜0.019g,二水合柠檬酸钠150g,六水合氯化铁3g,二水合氯化钙15.9g,七水合硫酸镁90g,硝酸铵90g,磷酸二氢钾90g,三水合磷酸氢二钾117.9g,乙酸钠541.41g,无菌水定容到300L。
其中,步骤2)中所述BG11培养基的配制方法如下:硝酸钠450g,磷酸氢二钾12g,七水合硫酸镁22.5g,二水合氯化钙10.8g,一水柠檬酸1.8g,柠檬酸铁铵1.8g,二水合乙二胺四乙酸二钠盐0.3g,碳酸钠6g混合后,加入微量元素储备液300mL,无菌水定容至300L。
其中,所述微量元素储备液的配制方法如下:硼酸2.86g,四水合氯化锰1.81g,七水合硫酸锌0.22g,二水合钼酸钠0.39g,五水合硫酸铜0.079g,六水合硝酸钴49.4mg,无菌水定容至1L。
其中,所述的突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1、所述的复合藻种或其制得的复合藻种土壤改良剂在修复重金属镉污染土壤中的应用也是本发明所提供的技术方案。
其中,所述应用包括改良农作物中的重金属镉污染;作为优选,每千克土壤添加至少2.5×108个突变型莱茵衣藻细胞;优选地,所述农作物为稻米。
与现有技术相比,本发明具有以下优点和有益效果:
1、本发明首次通过MNNG化学诱导方法对莱茵衣藻细胞突变,未经过转基因处理,不携带外源基因,生物安全性高,无额外的人工、设备和能源输入要求,生产成本低,再利用0.6mM氯化镉固体培养基定向筛选性状,获得了镉耐受藻株;该突变藻比表面积大,采用的是不易突变的真核生物,遗传性状稳定,能稳定地将有毒金属转化成无毒/低毒状态,对环境友好,不产生有害物质,对环境破坏小;能够进行土壤修复,可降低土壤重金属镉含量,高效降低稻米含镉量。
2、本发明首次将获得的镉耐受型突变藻株扩大培养,并混合小球藻,利用细胞浓缩技术制备成复合藻种土壤改良剂,该复合藻种土壤改良剂施至镉污染的稻田中,原位治理,相较于其他植物修复、电化学吸附及换土等方法减少了二次污染及运输成本,而且获得了远远优于单株藻降低土壤重金属镉含量的技术效果,大幅高效降低了稻米中含镉量。
附图说明
图1、本发明利用复合藻种修复土壤重金属镉污染的流程图;
图2、定向筛选的0.6mM氯化镉耐受型突变藻株与野生型藻株分别在滴片当天(D0)、第一天(D 1)、第三天(D 3)和第五天(D 5)生长情况对比图;
图3、对镉污染稻田进行不同方式的藻种处理后,稻米中的镉含量差异对比图;
图4、实施例3中步骤2实验地的环境温度记录情况。
具体实施方式
下面将对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明请求保护的范围。实施例中未注明具体条件者,均按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
本发明所述的重金属土壤改良剂的原材料为一株莱茵衣藻,拉丁学名:Chlamydomonas reinhardtii,购买于美国明尼苏达大学衣藻资源中心,编号为CC-1690wild type mt+,又名21gr,菌株网址为https://www.chlamycollection.org/product/cc-1690-wild-type-mt-sager-21-gr/。其属于绿藻门,绿藻纲,团藻目,衣藻科,衣藻属。莱茵衣藻为一种单细胞真核生物,椭球形,具有细胞壁;胞体前端有两条等长的鞭毛,可游动,鞭毛基部有伸缩泡;另外在细胞的近前端,有一个红色的眼点;叶绿体呈杯状,胞体内具有一枚淀粉核;细胞核位于细胞的中央偏前端,有的位于细胞中部或一侧。在生长旺盛时期主要进行无性繁殖,由单个细胞分裂产生2-16个游动的细胞,此外还可以进行有性生殖。
小球藻同属于绿藻门小球藻属单细胞淡水藻类,拉丁种名:Chlorella sp.,购买于中国科学院淡水藻种库,以下实施例所用小球藻编号:FACHB-10,菌株网址为http://algae.ihb.ac.cn/Products/ProductDetail.aspx?product=29。其在世界各地均有分布,以淡水水域中的种类较多,人工培养条件下能大量繁殖,不仅能光能自养,还能在异养条件下利用有机碳源快速繁殖。我国特有品种蛋白核小球藻中的蛋白质含量高,由于其营养价值丰富,常作为保健食品的原料。
实施例1突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1的获得
S1、配制TAP培养基:主要包括微量元素储备液、磷酸缓冲盐储备液、必需元素等。微量元素储备液以1L为例配制方法如下:称取乙二胺四乙酸二钠盐(C10H14N2Na2O8)50g,七水合硫酸锌(ZnSO4·7H2O)22g,硼酸(H3BO3)11.4g,四水合氯化锰(MnCl2·4H2O)5.06g,六水合氯化钴(CoCl2·6H2O)1.61g,五水合硫酸铜(CuSO4·5H2O)1.57g,四水合钼酸铵((NH4)6Mo7O24·4H2O)1.10g,七水合硫酸亚铁(FeSO4·7H2O)4.99g,氢氧化钾(KOH)20g,混合,使用ddH2O定容至1L;磷酸缓冲盐储备液以500mL为例配制方法如下:称取磷酸二氢钾(KH2PO4)28g,磷酸氢二钾(K2HPO4)54g,使用ddH2O定容到500mL;盐溶液以1L为例,配制方法如下:称取氯化铵(NH4Cl)20g,七水合硫酸镁(MgSO4·7H2O)5g,二水合氯化钙(CaCl2·2H2O)2.5g,混合,使用ddH2O定容到1L。TAP培养基配制方法如下:称取三羟甲基氨基甲烷(C4H11NO3)2.42g,再按顺序加入盐溶液10mL,磷酸缓冲盐储备液1mL,微量元素储备液1mL和冰醋酸1mL,使用ddH2O定容至1L,将配制好的TAP培养基121℃高压蒸汽灭菌20min,备用;
S2、灭菌完成后,降压冷却培养基,接入莱茵衣藻CC-1690连续光照曝气培养,初始浓度为5×105cells/mL,连续光照3天,至终浓度约为107cells/mL,进行诱变处理;
S3、在细胞对数期阶段,离心收集3×107个细胞,并用8mL含1ug/ml的MNNG化学诱变剂(购于MCE公司,货号HY-128612)的0.02M、pH为5.0的柠檬酸盐缓冲液重悬,置于25℃的避光条件下轻摇30min;将细胞离心收集后用100mL的0.02M、pH为5.0的柠檬酸盐缓冲液洗涤,用TAP培养基重悬细胞,同时制备未经MNNG处理的细胞作为对照,将诱导后的细胞在摇床上慢速、弱光、过夜恢复16h;
S4、收集细胞并用20%淀粉乳(每100mL无菌水中含20g淀粉)包裹,铺入含0.6mMCdCl2的固体培养基中,光周期(昼:夜=14:10)倒置培养10-14天,长出的即为耐受型突变体,将单克隆突变体再次点涂于0.6mM CdCl2的固体培养基中复筛得到稳定性状的镉耐受型突变型莱茵衣藻;
以上利用MNNG诱变处理莱茵衣藻细胞,获得镉敏感型突变体,其比例如表1所示:
表1 MNNG化学诱变剂处理莱茵衣藻野生型细胞获得的镉敏感型突变体数量统计表
将以上的耐受藻株全部挑选出来用TAP液体培养基活化得到藻细胞液体,分别滴106个藻细胞在0.6mM CdCl2的固体培养基中,得到耐受性最强的藻株,其分别在滴片当天、第一天、第三天和第五天的生长情况与图右列野生型藻株对比如图2所示。在其中选择镉耐受性最强的突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1,保藏于中国典型培养物保藏中心,保藏日期为2023年3月30日,保藏编号为CCTCC NO:M 2023436。
实施例2复合藻种土壤改良剂的制备
1、配制R培养基:其配制方法如下:称取硼酸(H3BO3)0.3g,七水合硫酸锌(ZnSO4·7H2O)0.3g,一水合硫酸锰(MnSO4·H2O)0.091g,六水合氯化钴(CoCl2·6H2O)0.06g,二水合钼酸钠(Na2MoO4·2H2O)0.06g,五水合硫酸铜(CuSO4·5H2O)0.019g,二水合柠檬酸钠(Na3C6H5O7·2H2O)150g,六水合氯化铁(FeCl3·6H2O)3g,二水合氯化钙(CaCl2·2H2O)15.9g,七水合硫酸镁(MgSO4·7H2O)90g,硝酸铵(NH4NO3)90g,磷酸二氢钾(KH2PO4)90g,三水合磷酸氢二钾(K2HPO4·3H2O)117.9g,乙酸钠(NaC2H3O2)541.41g,混合,然后使用无菌水定容到300L。将配制好的R培养基121℃高温灭菌20分钟;
2、灭菌完成后,冷却培养基,接入实施例1筛选出的衣藻镉耐受型突变藻株CCTCCNO:M 2023436无菌曝气培养,全光谱照明,光强3000lux,光周期时长昼:夜=14:10,温度22~27℃,使其终浓度达到107cells/mL;
3、配制BG11培养基,首先配制微量元素储备液:称取硼酸(H3BO3)2.86g,四水合氯化锰(MnCl2·4H2O)1.81g,七水合硫酸锌(ZnSO4·7H2O)0.22g,二水合钼酸钠(Na2MoO4·2H2O)0.39g,五水合硫酸铜(CuSO4·5H2O)0.079g,六水合硝酸钴(Co(NO3)2·6H2O)49.4mg,混合,加无菌水定容至1L。配制BG11方法如下:称取硝酸钠(NaNO3)450g,磷酸氢二钾(K2HPO4)12g,七水合硫酸镁(MgSO4·7H2O)22.5g,二水合氯化钙(CaCl2·2H2O)10.8g,一水柠檬酸(C6H10O8)1.8g,柠檬酸铁铵(FeC6H5O7·NH4OH)1.8g,二水合乙二胺四乙酸二钠盐(C10H14N2Na2O8·2H2O)0.3g,碳酸钠(Na2CO3)6g,混合,加入上述微量元素储备液300mL,使用无菌水定容到300L,高温灭菌20分钟;
4、灭菌完成后,待培养基冷却后接入小球藻无菌培养,全光谱照明,光强5000lux,光周期时长昼:夜=14:10,温度22~27℃,使其终浓度达到2×107cells/mL;
5、将衣藻、小球藻悬液分别离心浓缩,以细胞数量1:1配比混合,再浓缩至细胞总浓度为108cells/mL,无菌罐装即制成复合藻种土壤改良剂。
实施例3不同藻种分别处理及复合藻种处理镉污染土壤
1、不同藻种土壤改良剂的制备:分别将莱茵衣藻CC-1690、莱茵衣藻CCTCC NO:M2023436、小球藻FACHB-10通过细胞浓缩技术制成细胞总浓度为108cells/mL的藻液即得对应的野生型衣藻种土壤改良剂、突变型衣藻种土壤改良剂和小球藻种土壤改良剂;实施例2中制备得到的复合藻种土壤改良剂直接用。
2、本发明于2021年4月27日开始在湖南省株洲市渌口区进行镉污染稻田翻土,土壤中的初始镉含量0.711mg/kg,经度113°8'E,纬度为27°23'N,当地最高气温与最低气温如图4所示。5月14日在试验田中每亩施以藻种土壤改良剂(1013个藻细胞),同年5月30日试验田插秧、中间进行常规的田间管理,同年8月28日收割稻谷,处理后得到稻米。
5月14日在试验田中每亩施以藻种土壤改良剂的具体的施用方法如下:将藻种土壤改良剂处理分为五组:空白对照组(不进行任何处理,即图3中阴性对照)、野生型衣藻土壤改良剂(莱茵衣藻CC-1690)、突变型衣藻土壤改良剂(莱茵衣藻CCTCC NO:M 2023436)、小球藻土壤改良剂(小球藻FACHB-10)、实施例2制备的复合藻种土壤改良剂。
将每亩污染土壤施以藻种土壤改良剂为1013个藻细胞(对应于实施例2中的复合藻种土壤改良剂中的莱茵衣藻CCTCC NO:M 2023436和小球藻FACHB-10为各0.5×1013个藻细胞)换算为土壤的重量为每千克镉污染土壤施加5×108个藻细胞(对应于实施例2中的复合藻种土壤改良剂中的莱茵衣藻CCTCC NO:M 2023436和小球藻FACHB-10为各2.5×108个藻细胞)。
经过以上的藻种土壤改良剂处理后,该稻田中生长的稻米镉含量如表2和图3所示。
表2利用制得的藻种土壤改良剂分别处理镉污染稻田,收获的稻米中的镉含量(mg/kg)
相较于空白对照组,野生型衣藻藻株土壤改良剂和突变型衣藻藻株处理后的稻米镉含量依次递减,且呈现显著差异,其中突变型衣藻藻株处理组获得的稻米镉含量降至国家标准以下;小球藻处理组与空白对照组差异不明显,然而将复合藻种土壤改良剂处理土壤后其镉含量的降低大大超出了单独用两个藻种时的效果。
Claims (10)
1.一株突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1,其特征在于,所述突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1于2023年3月30日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023436。
2.一种复合藻种或其制得的复合藻种土壤改良剂,其特征在于,所述复合藻种或其制得的复合藻种土壤改良剂包括权利要求1所述的突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1。
3.根据权利要求2所述的复合藻种或其制得的复合藻种土壤改良剂,其特征在于,所述复合藻种或其制得的复合藻种土壤改良剂还包括小球藻(Chlorella sp.)FACHB-10。
4.根据权利要求3所述的复合藻种或其制得的复合藻种土壤改良剂,其特征在于,所述突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1与小球藻(Chlorella sp.)FACHB-10的细胞数量的比值为1:1~2:1,两者细胞总浓度为108 cells/mL。
5.权利要求2~4任一项所述的复合藻种或其制得的复合藻种土壤改良剂的制备方法,其特征在于,包括以下步骤:
1)将突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1接入灭菌好的R培养基中进行无菌曝气培养,全光谱照明,光强3000~5000 lux,光周期时长昼:夜=14:10,温度22~27℃,使其终浓度达到107 cells/mL;
2)将小球藻(Chlorella sp.)FACHB-10接入灭菌好的BG11培养基中进行无菌培养,全光谱照明,光强3000~5000 lux,光周期时长昼:夜=14:10,温度22~27℃,使其终浓度达到2×107 cells/mL;
3)将步骤1)得到的突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1的悬液和步骤2)得到的小球藻(Chlorella sp.)FACHB-10的悬液分别离心浓缩后以细胞数量1:1~2:1配比混合,再浓缩至两者的细胞总浓度为108 cells/mL,无菌罐装即制成复合藻种或复合藻种土壤改良剂。
6.根据权利要求5所述的复合藻种或其制得的复合藻种土壤改良剂的制备方法,其特征在于,每300L步骤1)中所述R培养基的配制方法如下:硼酸0.3g,七水合硫酸锌0.3g,一水合硫酸锰0.091g,六水合氯化钴0.06g,二水合钼酸钠0.06g,五水合硫酸铜0.019g,二水合柠檬酸钠150g,六水合氯化铁3g,二水合氯化钙15.9g,七水合硫酸镁90g,硝酸铵90g,磷酸二氢钾90g,三水合磷酸氢二钾117.9g,乙酸钠541.41g,无菌水定容到300L。
7.根据权利要求5所述的复合藻种或其制得的复合藻种土壤改良剂的制备方法,其特征在于,每300L步骤2)中所述BG11培养基的配制方法如下:硝酸钠450g,磷酸氢二钾12g,七水合硫酸镁22.5g,二水合氯化钙10.8g,一水柠檬酸1.8g,柠檬酸铁铵1.8g,二水合乙二胺四乙酸二钠盐0.3g,碳酸钠6g混合后,加入微量元素储备液300mL,无菌水定容至300L。
8.根据权利要求7所述的复合藻种或其制得的复合藻种土壤改良剂的制备方法,其特征在于,每1L所述微量元素储备液的配制方法如下:硼酸2.86g,四水合氯化锰1.81g,七水合硫酸锌0.22g,二水合钼酸钠0.39g,五水合硫酸铜0.079g,六水合硝酸钴49.4mg,无菌水定容至1L。
9.权利要求1所述的突变型莱茵衣藻(Chlamydomonas reinhardtii)MN1、权利要求2~4任一项所述的复合藻种或其制得的复合藻种土壤改良剂在修复重金属镉污染土壤中的应用。
10.根据权利要求9所述的应用,其特征在于,所述应用包括改良农作物中的重金属镉污染;作为优选,每千克土壤添加至少2.5×108个突变型莱茵衣藻细胞或者每亩重金属镉污染土壤施以至少含1013个藻细胞的复合藻种土壤改良剂;优选地,所述农作物为稻米。
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