CN114958819A - 一种稻田周丛生物的快速培养方法 - Google Patents
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Abstract
本发明提供一种稻田周丛生物的快速培养方法,采集稻田周丛生物与原位稻田土壤,利用原位稻田土壤制备土壤浸提液;将土壤浸提液与WC培养液按比例混合后用无氨水稀释,得到混合培养液;将载体分散于混合培养液中,在混合培养液中投加采集的稻田周丛生物作为微生物种源,进行周丛生物的培育。本发明的方法实现以廉价、绿色的方式快速培养生物量大、能够定殖成膜、群落结构稳定、活性高且生态功能性强的稻田周丛生物,为农业面源污染控制和农业高效生产的研究提供技术支持。
Description
技术领域
本发明涉及微生物培养领域,尤其涉及一种稻田周丛生物快速培养的方法。
背景技术
周丛生物,又称自然生物膜,是附着于水下任何基质表面的微生物聚集体,实质是一个完整的微生态系统,包括了微藻、真菌、细菌、原生动物和后生动物等生物组分,以及矿物质、胞外聚合物和碎屑等非生物组分。周丛生物在水体中分布极为广泛,在污水处理等污染防控中起着重要作用,同时也应用于环境监测等方面。
稻田中同样生长着大量的周丛生物,周丛生物具有固氮、解磷等功能,可以促进水稻生长。在生产实践中,施放的肥料不可能在整个生长期都符合水稻的养分需求曲线,过量与不足的情况都存在。尤其是过量时,多出来的氮、磷等养分往往流失到水体和大气中,造成污染。这时,周丛生物就像仓库一样将部分氮、磷吸收和储存起来,能有效减少污染。
众多学者开展了对周丛生物(或类似的自然生物膜)的研究,但野外采样后的周丛生物如何在实验室内快速培养成为了一个难题。在已有的专利中,周丛生物的培养方法各有不同:
专利CN102559498A公开一种用于培养微生物的营养剂及微生物培养方法,该方法驯化出的微生物为污水处理过程中所需要的微生物,并不适用于稻田周丛生物的培育。专利CN108410736A公开一种微生物培养方法,该方法主要用于批量培养微生物,该方法利用培养液输送管做到及时的对于培养罐内的培养液及时的补充,最大限度提高微生物的繁殖和生长,并利用搅拌板的自身发电提供培养罐内的适宜温度。但该方法需要特殊的培养罐,提高了实验装置成本。专利CN110564628A公开一种全营养基质微生物培养方法,该方法利用马铃薯中的淀粉和蛋白质等营养物质,虽然培养出的菌丝体健壮、浓密、旺盛,但该培养基的营养源与稻田环境差异太大,可能导致周丛生物的群落结构和优势菌群等方面出现较大差异。专利CN111304087A公开一种微生物培养方法及提取工艺,该培养方法采用的是现有的实验室微生物培养方式,存在培养时间长,培育量小等不足。
综上所述:目前稻田周丛生物的培养方法主要存在以下几点不足,一是培养条件需要特殊的装置或设备,为实验提高了培养成本;二是培养时间长、培育微生物量小,活性不强、培养的周丛生物群落结构单一,且性质不稳定,易造成脱落的现象,不能满足实验或工程需求;三是现有的培养方法导致稻田周丛生物生长为絮状,形态与田间周丛生物有较大差异,部分周丛生物无法定殖成膜,无法形成稳态的微型生态系统。因此,探究快速培育生物量大、能够定殖成膜、群落结构稳定的稻田周丛生物的方法,是本发明的立足点。
发明内容
本发明的目的在于针对上述现有培养技术存在的不足,提供一种快速培养生物量大、能够定殖成膜、群落结构稳定的稻田周丛生物的方法。该方法旨在以廉价、绿色、简单易行、快速的方法培育生物量大,群落结构复杂的稻田周丛生物,为农业面源污染控制和农业高效生产的研究提供技术支持。
为实现上述技术目的,本发明采用如下方案:
一种稻田周丛生物的快速培养方法,包括:
采集稻田周丛生物与原位稻田土壤,利用原位稻田土壤制备土壤浸提液;
将土壤浸提液与WC培养液按比例混合后用无氨水稀释,得到混合培养液;
将载体分散于混合培养液中,在混合培养液中投加采集的稻田周丛生物作为微生物种源,进行周丛生物的培育。
作为一种优选的实施方式,所述混合培养液的组成以体积含量计,包括9~11%土壤浸提液、0.95~1.05%WC培养液,其余为无氨水。
作为一种优选的实施方式,所述周丛生物培育的温度为29.5~31.5℃。
作为一种优选的实施方式,以湿重计,所述稻田周丛生物投加浓度为1.0~2.0g/L,投加的周丛生物含水率为39%~47%。
作为一种优选的实施方式,周丛生物培育时,在混合培养液承装容器上封盖一层聚乙烯膜,并扎10~15个小孔。
作为一种优选的实施方式,周丛生物培育时,每5天补充混合培养液以至初始体积;如需持续培养,周丛生物定殖成膜后,每10天更换一次混合培养液。
作为一种优选的实施方式,所述土壤浸提液的制备方式为:
将采集的原位稻田土壤去杂后,浸泡于无氨水中震荡混合,之后离心取上清液,即为土壤浸提液。
作为一种优选的实施方式,去杂的原位稻田土壤与无氨水按照1g:10mL的质量体积比混合。
作为一种优选的实施方式,还包括回收周丛生物:在周丛生物培育完成后,从载体上回收附着的周丛生物,以及将混合培养液离心分离,沉淀物为周丛生物。优选的,离心温度为25±1℃,转速为7000~8000rpm,时间为8~10min。
本方法的有益效果在于:
1、本发明在使用土壤浸提液和WC混合培养液,为周丛生物提供了更为接近原位的培养条件,为一部分在传统培养方法中无法存活的微生物提供了生存条件。
2、无需特殊的实验装置,通过对周丛生物生存环境的精确控制,可以廉价、绿色、快速的在实验室培育出大生物量的周丛生物;且培养出的周丛生物能够快速定殖成膜、群落结构稳定、活性高且生态功能性强。
3、本发明操作简便易行,便于观察,可灵活对不同时期、不同条件下的周丛生物进行测定,极大降低了实验室的工作强度;
4、本发明中聚氨酯载体不仅为周丛生物生长提供了附着条件,还有益于周丛生物的回收,省略了离心和过滤工序,节省了人工与设备投入成本。
附图说明
图1-周丛生物在不同培养条件下的生物量。
图2-快速培养与传统培养的pH变化。
图3-快速培养与传统培养的OD600值变化。
图4-快速培养与传统培养的OD680值变化。
具体实施方式
下面结合附图说明和具体实施方式对本发明的技术方案作进一步阐述。
实施例使用的技术方案包括如下步骤:
S1:周丛生物的扩大培养:以江苏镇江句容采集的稻田周丛生物为微生物种源;以原位稻田土壤为制备土壤浸提液原材料;
将采集的稻田土壤去除异物如植物根茎、树叶和大块石硕后,按照土样与无氨水1g:10mL的比例称取土壤样品,土样浸泡在无氨水中,用恒温振荡箱以250rpm振荡2h。再将浸提液以2500r/min的转速离心5min,降低土壤浸提液浊度,取上清液为土壤浸提液。
所述WC培养液组分为:NaNO3 85.1mg·L-1、KH2PO4 8.71mg·L-1、MgSO4·7H2O36.97mg·L-1、NaHCO3 12.6mg·L-1、CaCl2·2H2O 36.76mg·L-1,NaSiO4·9H2O 28.42mg·L-1、H3BO3 24mg·L-1、微量元素溶液Ⅰ1ml·L-1、VB12溶液1ml·L-1、VB1溶液1ml·L-1、生物素溶液1ml·L-1;
微量元素溶液Ⅰ的组成:Na2EDTA·2H2O 4.36g·L-1、FeCl3 3.15g·L-1、ZnSO4·7H2O 22g·L-1、MnCl2·4H2O180g·L-1、CuSO4·5H2O 2.5g·L-1,NaMoO4·2H2O 6.3g·L-1、Na3VO4 18g·L-1;
VB12溶液的组成:HEPES缓冲液12g·L-1、VB12 0.135g·L-1;
VB1溶液的组成:HEPES缓冲液12g·L-1、VB12 0.335g·L-1;
生物素溶液的组成:HEPES缓冲液12g·L-1、生物素0.025g·L-1;
三种维生素溶液配制在200mL HEPES缓冲液中调节pH至7.8,并低温避光保存。
将土壤浸提液与WC培养液成不同比例混合,其混合液为培养基;将聚氨酯(长:5cm;宽:5cm;厚度:1cm)载体分散于培养液中,作为周丛生物附着载体。培养系统在智能光照培养箱中,光照强度为10000~12000LUX,光照/黑暗状态为12h/12h;在一个温度梯度下培养周丛生物;每5天向培养系统中补充混合培养液,保持初始体积不变。如需持续培养,周丛生物定殖成膜后,每10天更换一次混合培养液;培养周期为30d,定期检测所需指标。
S2:周丛生物的回收:部分周丛生物附着于载体,可直接回收;将步骤S1中的培养液离心分离,沉淀物为周丛生物。
具体实施时,在步骤S1中,为了模拟稻田环境,在传统培养方法基础上,经查阅文献及现场调研,了解培养稻田周丛生物的生长温度等培养条件。经查阅文献确定土壤浸提液比例;在传统培养的基础上确定WC培养液比例范围;经现场调研,根据水稻生长时当地的气候,确定培养温度范围。
采用响应面分析方法设计实验,设定土壤浸提液比例范围为0~20%(体积含量);WC培养液比例范围为0.5~1.5%(体积含量);培养温度范围为26~35℃。
步骤S1中实验组实验设计参数如下:
注:表中-1为设定范围的最小值,0为设定范围的中间值,1为设定范围的最大值。
具体实施时:在步骤S1中,在周丛生物扩大培养时,周丛生物投加浓度为1.0~2.0g(湿重)/L,周丛生物含水率为39.44%~47.04%。在扩大培养时,周丛生物投加浓度越高,培养效果越好,但增加投加浓度,同样会增加成本。在具体实施时依据实际情况,周丛生物投加浓度在1.0~2.0g(湿重)/L,周丛生物含水率为39%~47%。具体实施时,含水率可以通过离心分离、过滤等方式控制;也可以根据本专利中的数据将周丛生物转换为干重,以干重来衡量周丛生物添加量。
具体实施时,在S1步骤中,培养系统需封盖一层聚乙烯膜,扎10~15个小孔,在保证周丛生物生长需求的同时,避免外来微生物污染样品,以及减少培养液通过蒸发等过程的损失。
具体实施时,在步骤S2中,离心温度为25±1℃,转速为7000~8000rpm,时间为8~10min。
下面结合附图和具体实施方式对本发明作进一步详细说明。以下实施例仅用于更加清楚地说明本发明的技术方案,而不能以此来限制本发明的保护范围。
实施例1
实施例1对上诉步骤S1中的培养30d后的13个实验组进行生物量的比较。
利用Design Expert 10.0.1软件对所得的结果进行逐步回归,确定快速培养参数为土壤浸提液比例9~11%、WC培养液比例0.95~1.05%、培养温度29.5~31.5℃,此时周丛生物的生物量增长率高,微生物群落结构稳定,扩培效果最佳。具体实施时,快速培养为组13,传统培养为组5。
取0.2g(湿重)稻田周丛生物,控制光照强度为12000LUX,光照/黑暗状态为12h/12h,培养时间为30d。持续检测实验组的pH、OD600与OD680,设计13组培养实验,如表1所示(体系中液体总体积为200mL)。
培养时间结束后,回收周丛生物,测量生物量。
表中生物增长率为相比第5组增长率,第5组为传统培养参数。
表1 13组培养实验生物量增长率结果对比。
组别 | 土壤浸提液 | WC培养液 | 培育温度 | 生物量增长率 |
1 | 0 | 0.5% | 30.5℃ | 34% |
2 | 20% | 0.5% | 30.5℃ | 167% |
3 | 0 | 1.5% | 30.5℃ | 98% |
4 | 20% | 1.5% | 30.5℃ | 215% |
5 | 0 | 1% | 26℃ | - |
6 | 20% | 1% | 26℃ | 74% |
7 | 0 | 1% | 35℃ | 86% |
8 | 20% | 1% | 35℃ | 165% |
9 | 10% | 0.5% | 26℃ | 22% |
10 | 10% | 1.5% | 26℃ | 52% |
11 | 10% | 0.5% | 35℃ | 45% |
12 | 10% | 1.5% | 35℃ | 118% |
13 | 10% | 1% | 30.5℃ | 249% |
实施例2
实施例2是周丛生物扩培过程中快速培养与传统培养的pH变化对比。
培养过程中,pH的改变曲线和微生物生长曲线有关,从培养开始,随着微生物繁殖增加,代谢产物累积加快,培养基中的pH也呈现较快速度的改变。
用pH计4天一次连续测定培养过程中的pH变化(图2)。快速培养在0~9天pH值从初始7.3提高至9.7,在此期间周丛生物快速繁殖,定殖成膜;传统培养在0~13天从初始7.2提高至10,其中0~5天生长缓慢。快速培养在第13天达到pH的最大值后,培养系统自我调节,pH下降并保持在适宜生长的范围;而传统培养13~21天都保持在一个较高的pH范围,过高的pH环境不利于周丛生物的快速繁殖(图2)。
实施例3
实施例3是周丛生物扩培过程中快速培养与传统培养的OD600值变化对比。
OD600是将波长设定为600nm时测定的光密度值,是追踪液体培养物中微生物密度的标准指标,通常用来指示菌体细胞密度。
每2天一次连续测定培养过程中的OD值(图3)。快速培养在0~13天OD600值快速上升至最大值,培养系统中的周丛生物快速繁殖,而后在胞外聚合物等物资的作用下快速团聚,定殖成膜,培养液中的菌体细胞密度快速下降,OD600值下降;传统培养在0~19天OD600值较缓上升至最大值,25天后,培养系统部分周丛生物呈絮状,悬浮于培养系统中,无法形成稳定的生物膜,培养液中的菌体密度无法达到一个理想的较低水平(图3)。
实施例4
实施例4是周丛生物扩培过程中快速培养与传统培养的OD680值变化对比。
OD680就是Optical Density at 680nm,也就是680nm波长处的光密度,也称为光吸收值。这一吸收主要是由于藻类细胞中的叶绿素a的存在,通过OD680可以大致估算藻类细胞密度、生物量等。
OD680值的变化与OD600值的变化拥有相似的规律,不同之处在于快速培养第7天达到最大值,第17天时周丛生物已经达到第二次发育峰值,比传统培养第19天才达到第一次生长最大值要生长快速(图4)。
Claims (10)
1.一种稻田周丛生物的快速培养方法,其特征在于,包括:
采集稻田周丛生物与原位稻田土壤,利用原位稻田土壤制备土壤浸提液;
将土壤浸提液与WC培养液按比例混合后用无氨水稀释,得到混合培养液;
将载体分散于混合培养液中,在混合培养液中投加采集的稻田周丛生物作为微生物种源,进行周丛生物的培育。
2.根据权利要求1所述的快速培养方法,其特征在于,所述混合培养液的组成以体积含量计,包括9~11%土壤浸提液、0.95~1.05%WC培养液,其余为无氨水。
3.根据权利要求1所述的快速培养方法,其特征在于,所述周丛生物培育的温度为29.5~31.5℃。
4.根据权利要求1所述的方法,其特征在于,以湿重计,所述稻田周丛生物投加浓度为1.0~2.0g/L,投加的周丛生物含水率为39%~47%。
5.根据权利要求1所述的方法,其特征在于,周丛生物培育时,在混合培养液承装容器上封盖一层聚乙烯膜,并扎10~15个小孔。
6.根据权利要求1所述的方法,其特征在于,周丛生物培育时,每5天补充混合培养液以至初始体积;如需持续培养,周丛生物定殖成膜后,每10天更换一次混合培养液。
7.根据权利要求1所述的方法,其特征在于,所述土壤浸提液的制备方式为:
将采集的原位稻田土壤去杂后,浸泡于无氨水中震荡混合,之后离心取上清液,即为土壤浸提液。
8. 根据权利要求7所述的方法,其特征在于,去杂的原位稻田土壤与无氨水按照1 g:10 mL的质量体积比混合。
9.根据权利要求1所述的方法,其特征在于,还包括回收周丛生物:
在周丛生物培育完成后,从载体上回收附着的周丛生物,以及将混合培养液离心分离,沉淀物为周丛生物。
10. 根据权利要求9所述的方法,其特征在于,离心温度为25±1℃,转速为7000~8000rpm,时间为8~10 min。
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