CN114956348A - 一种利用自养处理沼液废水后的微藻治理芋艿连作障碍的方法 - Google Patents
一种利用自养处理沼液废水后的微藻治理芋艿连作障碍的方法 Download PDFInfo
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Abstract
本发明提供一种利用自养处理沼液废水后的微藻治理芋艿连作障碍的方法,即利用微藻净化养猪沼液废水,然后在将处理废水后的微藻藻液来施加到芋艿连作土壤中,来改善土壤微生物群落结构,促进土壤营养循环,治理作物连作障碍。本发明利用活性微藻治理芋艿连作障碍,探究奉化芋艿连作障碍原因,针对芋艿连作障碍特征因子筛选营养价值高且耐污能力强的藻株,利用活性微藻处理农村养殖污水,并基于微藻特定的活性功能物质与丰富的矿物营养,改善芋艿连作土壤,提升芋艿自身抗性,以期为治理芋艿连作障碍及改善农村生态环境提供可行性技术支持。
Description
技术领域
本发明属于微藻应用技术领域,具体涉及一种利用自养处理沼液废水后的微藻治理芋艿连作障碍的方法。
背景技术
芋,又称芋艿,学名Colocasia esculenta(L.)Schott,属天南星科芋属的多年生单子叶草本湿生植物。它广泛分布于亚洲及大洋洲热带和部分亚热带地区,是世界上食药两用的重要粮食作物和经济作物之一。奉化芋艿因具有肉质细腻、粉质,无明显红筋,口感糯滑,风味独特,营养丰富等特点,而深受广大消费者的青睐,是浙江省以至全国闻名的特色农产品。奉化芋艿的主要成分是淀粉、粗蛋白、氨基酸及维生素C、B1、B2和维生素D等。传统医学认为,芋性辛、甘平,入胃、肠,可通便散结、补中益气,具有营养保健功效以及较高的商品经济价值。
芋艿连续在同一块地上种植2年及以上会导致植株矮小、球茎变小,产量降低,且出现“煮不烂”等连作障碍现象,严重影响经济效益,是限制奉化芋艿产业发展的主要瓶颈。
传统轮作、抗病品种选育、嫁接育苗、土壤消毒、生物强还原等虽能在一定程度上缓解连作障碍,但存在选育时间长、品质改变、化学试剂残留等问题,仍然缺乏针对特定障碍因子的绿色生态防治技术。目前关于芋艿连作障碍的防治研究也很少报道。农作物的连作障碍发生涉及诸多因子,而且与作物自身类型及特性密切相关。一般而言,作物连作障碍的发生主要有以下3个重要原因:
①土壤养分不均衡及土壤理化性质恶化;
②土壤病原菌累积和根际微生态功能失调;
③次生代谢产物化感物质等引起毒性作用。
因此,经济有效的增强农作物自身抗性与生态健康的改良土壤肥力及微生物区系可能是绿色生态治理芋艿连作障碍的两大关键措施。
发明内容
本发明的目的是提供一种利用自养处理沼液废水后的微藻治理芋艿连作障碍的方法,即利用微藻净化养猪沼液废水,然后在将处理后的微藻藻液来施加到芋艿连作土壤中,进一步改善土壤微生物群落结构,促进土壤营养循环,治理作物连作障碍,从而弥补现有芋艿连作障碍治理技术的不足。
本发明首先提供一种利用自养处理沼液废水的方法,所述的方法,是使用微藻种子液接种到沼液废水中来处理;
所述的微藻,作为优选,为螺旋藻和/或小球藻;
所述的微藻种子液,是将微藻接种到培养基中,在25℃,模拟室外光照条件,具体为光照强度3000-4000lux,光照周期12h:12h条件下培养获得的。
本发明另一个方面还提供一种利用处理沼液废水后的微藻治理芋艿连作障碍的方法,是使用处理沼液废水后的微藻藻液,制成藻液后施加到用于连作芋艿的土壤中;
所述的微藻,优选为螺旋藻和/或小球藻;
所述的沼液废水,为养猪过程中产生的沼液废水。
本发明利用活性微藻治理芋艿连作障碍,探究奉化芋艿连作障碍原因,针对芋艿连作障碍特征因子筛选营养价值高且耐污能力强的藻株,利用活性微藻处理农村养殖污水,并基于微藻特定的活性功能物质与丰富的矿物营养,改善芋艿连作土壤,提升芋艿自身抗性,以期为治理芋艿连作障碍及改善农村生态环境提供可行性技术支持。
附图说明
图1为本发明所述不同藻类的生长曲线图,其中横坐标为培养时间(d),纵坐标为吸光度
图2中图a为本发明所述筛选微藻处理猪废水的生物量图,其中横坐标为培养时间(d),纵坐标为生物量(g/L),图2b为本发明所述筛选微藻处理猪废水的生物产率图,其中横坐标为微藻种类,纵坐标为生物量产率(g/L/d);
图3a为致病真菌种类图,图3b为有益真菌种类图,其中KL表示空白连作,LL表示螺旋藻连作。
具体实施方式
本发明将筛选的优势藻株处理养猪沼液废水,然后用处理沼液废水后的微藻液体来灌溉发生连作障碍的芋艿田,使土壤肥力与微生物群落结构得到改善并抑制芋艿连作障碍病原因子,提高芋艿品质,进而为改善农村养殖生态环境,提高设施农业品质与经济发展,以及农村生态环境保护提供生态资源化技术支持。
本发明使用的BG11培养基配方如下:K2HPO4 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,EDTANa2 0.001g/L,NaNO31.5g/L,Na2CO30.02g/L,A5微量元素液1.0ml/L,用1M HCl调整pH为7.0。其中A5微量元素液组成:H3BO3 2.86g/L,MnCl2·4H2O 1.86g/L,ZnSO4·7H2O 0.22g/L,Na2MoO4·2H2O 0.39g/L,CuSO4·5H2O 0.08g/L和Co(NO3)2·6H2O 0.05g/L;
SP培养基配方为:NaHCO3 13.61g/L,Na2CO3 4.03g/L,K2HPO4 0.50g/L,NaNO32.50g/L,K2SO4 1.00g/L,NaCl 1.00g/L,MgSO4·7H2O0.20g/L,CaCl2·2H2O 0.04g/L,FeSO4·7H2O 0.01g/L,A5微量元素液1.0ml/L,其中A5微量元素液组成:H3BO3 2.86g/L,MnCl2·4H2O1.86g/L,ZnSO4·7H2O 0.22g/L,Na2MoO4·2H2O 0.39g/L,CuSO4·5H2O0.08g/L和Co(NO3)2·6H2O 0.05g/L。
下面结合实施例和附图对本发明进行详细的描述
实施例1:利用微藻自养处理沼液废水
(1)优势藻株的筛选与鉴定
对4株微藻藻株处理养猪沼液废水的效果进行筛选,其中小球藻(Chlorellavulgaris),栅藻(Scenedesmus dimorphus),极大螺旋藻(Spirulina platensis),丝状藻(Filamentous oleaginous)购买自中国科学院武汉水生生物研究所淡水藻种库,另外2株黄丝藻(Tribonema sp.)和集胞藻(Synechocystis sp.)来自本实验室前期筛选保藏。分别采用BG11培养基和SP培养基,在培养温度为25℃,光照强度为3000-4000lux,光照周期12h:12h条件下培养微藻。
(2)藻株处理养猪沼液废水的效果测定
A、微藻的生物量测定:
通过重量分析法测试选定的微藻生物量浓度(DW,g/L)。具体而言,将0.45μm(直径50mm)的GF/C滤膜(Whatman,England)煮沸三次,然后在105℃的烘箱中加热至恒重(DW0,mg),定量10mL体积的藻液并倒入抽滤器中,抽滤至称重的滤膜上;将含有藻细胞的膜在105℃烘箱干燥6-10小时,然后测量干重(DW1,mg)。DW计算如下:
DW=(DW1-DW0)/10
其中10代表每个样品的体积(mL)。
生物量产率(Pbiomass)通过以下等式计算:
其中X0是微藻生物量初始浓度(g/L),X是微藻生物量最终浓度(g/L),t是以天为单位的培养持续时间(d)。
B、微藻的采收及生化组分分析:
为了获取生物量,将培养物静置并分离上清液。在西格玛6-16KS离心机(美国西格玛奥尔德里奇)中,生物量在4℃和10000×g(RCF)下离心。离心时间根据微藻的需要而不同,从10-30min不等。收集的部分新鲜生物量在冷冻干燥机(美国赛默飞世尔科学公司)中冷冻干燥。新鲜和冷冻干燥的生物质均在-18℃保存,使用冷冻干燥的微藻生物质作为样本。
a)油脂含量
藻类细胞的总脂质含量通过改进的氯仿-甲醇法测定,称取约25mg(W1)微藻粉,加入烘干的石英砂100mg,加入2.5mL甲醇和1.25mL氯仿。高速震荡5min。摇床12h,离心(9000rmp,10min)取出上清至新管1。向沉淀管中再加入2.5mL甲醇,1.25mL氯仿,高速震荡5min,放摇床2h,离心取上清至新管1。向新管1中加入2.5mL氯仿和4.5mL1%NaCl,保证最终体系为甲醇:氯仿:1%NaCl=2:2:1.8,震荡混匀。将新管1于8000rmp离心10min,去上清液,下层液转移入20mL干净玻璃管(已称重W2)。在61℃下吹氮气约10min后,蒸发氯仿,在105℃下干燥3h,冷却并称重(W3)。
总脂质含量(%)=(W3-W2)/W1·100%
b)多糖含量
微藻的多糖含量通过苯酚-硫酸法测定,准确称取藻粉10mg,加入pH=5的蒸馏水1mL,超声30min,加入木瓜蛋白酶和纤维素酶(1:1)0.5mL,40℃酶解30min,沸水灭酶10min,用70℃热水浸提4h,离心(9000r,15min,0℃),取上清液入15mL离心管,添加3倍体积95%乙醇(4.5mL)静置过夜(4℃),离心(9000r,15min,0℃),倒掉上清液,加入2mL纯水后震荡充分溶解(反应10min),离心(9000r,20min,4℃);加入等体积预冷的10-20%三氯乙酸(TCA)溶液,立刻混匀,冰上放置30min,离心(9000r,20min,4℃),取上清液,则为除蛋白多糖。然后分别吸取0、0.05、0.1、0.15、0.2、0.25、0.3mL的标准葡萄糖溶液(0.1mg/mL)置于离心管(编号0-6)中,加蒸馏水补至0.5mL。向管中分别添加0.25mL 6%苯酚溶液和1.25mL浓硫酸,自然条件下反应5min并经过沸水浴20min后冰水浴10min,在490nm处测定吸光度。分别以葡萄糖浓度、吸光度值为横纵坐标,制作葡萄糖标准曲线,进而计算样品中多糖的含量。
c)蛋白质含量
藻类细胞的总蛋白质通过考马斯亮蓝G250法测定。准确称量冻干微藻藻粉样品,倒入加了研磨珠的研钵中,用液氮研磨至粉末状。分析过程如下:添加蛋白质提取物(50mMTris–HCl(pH=7.4)、150mM NaCl、1mM PMSF、1mM EDTA、1%Triton X-100)。在冰上放置4h,以12000rpm的转速离心20min,提取上清液,并完成样品制备。将样品稀释20-40倍,取样品液0.5mL,然后加入2.5mL考马斯亮蓝试剂,震荡混匀,静置2min后于595nm测定吸光度。按照梯度稀释0.5mg/mL牛血清蛋白溶液进行标准曲线的制作,根据标准曲线即可计算出样品的浓度。
C、废水理化特征分析
原始养猪沼液废水收集自浙江省宁波市奉化区的生猪养殖厂。在实验之前,通过沉淀和过滤对原猪废水进行预处理,以去除颗粒固体。上清液在用于微藻培养之前储存在4℃下。预处理废水的pH值约为5.4,猪废水预处理后的COD、氨氮(NH3-N)和总磷(TP)的原始平均浓度为4817.3mg/L,478.6mg/L和53mg/L。
利用藻株处理沼液废水(pH调整至7.5左右),培养时间12天,分别测定藻细胞生长及废水中NH3-N、TP、COD的污染特征变化。
根据Cheng等人(2020年)的方法分析理化特性。在培养周期结束后,收集6.0mL样品于离心管中,并以6000×g离心10min。根据Hach DR 5000分光光度计手册(Hach,2008),适当稀释上清液,以分析氨氮(NH3-N)、总磷(TP)和化学需氧量(COD)。
表1:藻类活性物质含量及其对沼液废水污染物的去除效率
由表1可知,两株优选微藻在处理养猪沼液废水后,细胞活性物质的含量有差异变化。其中,螺旋藻(Spirulina platensis)的蛋白和多糖的含量显著高于小球藻(Chlorellavulgaris),而脂质含量则相反。在沼液废水污染物去除效率方面,小球藻对氨氮(NH3-N)、总磷(TP)和化学需氧量(COD)的去除效率略高于螺旋藻。
实施例2:微藻治理芋艿连作障碍
处理完沼液废水后的微藻使用气浮方法采收,排除废水中其他物质的可能影响,采收后的藻泥用自来水悬浮制备成1.5g/L的螺旋藻藻液和小球藻藻液,分装在塑料容器里。
选取浙江省宁波奉化滕头生态村萧王庙街区某芋艿连作障碍田(北纬29°25′~29°47′,东经121°03′~121°46′)为试验田。设计采用定位连作与非连作对比设置,芋艿设连作和非连作2种处理,同一块地一半连作,另一半非连作。设置连作和非连作第一垄为空白对照,第二垄为螺旋藻藻液灌溉。第三垄为小球藻藻液灌溉。
将处理养猪沼液废水后的微藻藻液灌溉芋艿植株(分别在幼苗期、发棵期灌溉2次),采用5点取样的方法,在芋艿成熟期每个小区随机选取5株,测量芋艿株高、叶长和叶宽,并在植株主茎周围20cm取0~10cm土层的根际土壤,除去石头、虫体、草枝等杂物后,多点土壤样品充分混合,采用四分法留取土样。处理后的样品立刻用液氮处理,随即使用干冰保存,运输至实验室,-80℃冷冻保存,用于土壤微生物多样性分析。
实验材料委托北京诺禾致源科技股份有限公司进行高通量测序。使用Uparse软件(v7.0.1001)对所有样本进行OTUs聚类,用Qiime软件(Version 1.9.1)进行物种注释分析和阿尔法(Alpha)多样性分析,使用R软件(Version 2.15.3)进行Alpha多样性指数组间差异分析和Beta多样性指数组间差异分析。根据样品在数据库中的功能注释及丰度信息,选取丰度排名前35的功能及它们在每组样品中的丰度信息绘制热图,并从功能差异层面进行聚类,得到结论:加入螺旋藻后连作(LL)芋艿根际土壤病原菌(Plant Pathogen)丰富度显著降低,而土壤有益菌(Ectomycorrhizal)丰度显著提高。进一步从数据库中筛选出根系病原菌和有益菌的OTU数目,分别绘制聚类丰度热图。结果如图3所示。
从图中可清晰的看出,芋艿根际土壤中真菌病原菌和有益菌丰度排名较大的物种。图3a图表示土壤病原菌在LL和KL中丰度的变化情况,其中与空白连作(KL)相比,在螺旋藻连作(LL)中Curvularia、Erysiphe、Clonostachys、Synchytrium、Gaertneriomyces等病原菌丰度下降十分显著。图3b图表示土壤有益菌在LL和KL中丰度的变化情况,与空白连作(KL)相比,在螺旋藻连作(LL)中Ceratobasidium、Pulveroboletus等有益菌丰度提高非常显著。
在芋艿采收期,对植株株高、叶长和叶宽进行测量,结果如表2所示。
表2:芋艿植株的生长特征数据表
从表2可以看出,芋艿非连作、连作两组样本的植株高和叶面积差异显著,非连作样品植株高和叶面积均显著大于连作样品;与空白对照相比,加入小球藻、螺旋藻藻肥后,芋艿植株株高、叶长和叶宽均不同幅度的显著提高,说明螺旋藻、小球藻均能改善芋艿植株的生长特征。
Claims (7)
1.一种利用自养处理沼液废水的方法,其特征在于,所述的方法,是使用微藻种子液接种到沼液废水中来发酵处理沼液废水。
2.如权利要求1所述方法,其特征在于,所述的微藻为螺旋藻和/或小球藻。
3.如权利要求1所述方法,其特征在于,所述的微藻种子液,是将微藻接种到培养基中,在25℃,光照强度为3000-4000lux,光照周期12h:12h条件下培养获得的。
4.如权利要求3所述的方法,其特征在于,所述的培养基为BG11培养基或SP培养基。
5.一种治理芋艿连作障碍的方法,其特征在于,所述的方法是使用微藻处理养猪沼液废水,然后收集微藻,制成藻液后施加到用于连作芋艿的土壤中。
6.如权利要求5所述的方法,其特征在于,所述微藻为螺旋藻和/或小球藻。
7.如权利要求5所述的方法,其特征在于,所述的沼液废水为养猪过程中产生的沼液废水。
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