WO2021212919A1 - 一种基于绝迹稀释培养组的功能微生物高通量分析和选育方法 - Google Patents

一种基于绝迹稀释培养组的功能微生物高通量分析和选育方法 Download PDF

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WO2021212919A1
WO2021212919A1 PCT/CN2020/141959 CN2020141959W WO2021212919A1 WO 2021212919 A1 WO2021212919 A1 WO 2021212919A1 CN 2020141959 W CN2020141959 W CN 2020141959W WO 2021212919 A1 WO2021212919 A1 WO 2021212919A1
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dilution
species
extinct
culture group
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许玫英
邓通初
郭俊
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广东省微生物研究所（广东省微生物分析检测中心）
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  • the invention belongs to the technical field of microorganisms, and specifically relates to a method for high-throughput analysis and breeding of functional microorganisms based on an extinct dilution culture group.
  • Culturomics is a culture technology that cultivates target microorganisms through a variety of culture conditions, and uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and 16S rRNA gene sequencing technology to carry out high-throughput identification of them. .
  • MALDI-TOF-MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • 16S rRNA gene sequencing technology 16S rRNA gene sequencing technology
  • the application of the culture group to the study of microbial ecology will form an effective complement to non-cultivation technologies such as high-throughput sequencing technology, and more accurately clarify the problems in microbial ecology.
  • the culture group technology is characterized by high-throughput identification of microorganisms, and for special groups of microorganisms in the environment, it is still necessary to combine different culture methods to separate different microorganism species.
  • the purpose of the present invention is to provide a high-throughput analysis and breeding method of functional microorganisms based on extinct dilution culture groups, which can effectively separate species with different abundance and different functional characteristics in the environment.
  • the method for high-throughput analysis and breeding of functional microorganisms based on the extinct dilution culture group of the present invention includes the following steps:
  • step d Collect the bacteria in the sample after step c to determine the potential activity, extract the total DNA, and amplify the V4-V5 region of the 16S rRNA gene for high-throughput sequencing, and analyze the microbial community of the extinct dilution culture group using bioinformatics methods Species composition, and its relationship with potential active characteristics;
  • the samples According to the characteristics of the microbial community composition of the extinct dilution culture group samples, select the samples first to use solid enrichment medium to separate and screen the target functional species for single colony, purify, and perform species identification and functional identification to verify whether the target functional species has been isolated .
  • the selective medium containing the substrate and the determination of the concentration change of the substrate in the selective medium are determined according to the function of the screening target microorganism.
  • Na 2 S can be used as the substrate to screen for nitrate reduction.
  • Sulfur-oxidizing bacteria, nitrate-reducing sulfur-oxidation mediated by nitrate-reducing sulfur-oxidizing bacteria is an important process that couples the nitrogen cycle and the sulfur cycle.
  • the concentration of nitrate, nitrite, and sulfate can be determined by ion chromatography, as well as biological
  • NaS 2 O 3 can also be used as a substrate to screen bacteria that oxidize thiosulfate.
  • Thiosulfate is an important intermediate product in the sulfide oxidation process and sulfur cycle.
  • the environmental sample can be soil, sediment, water and other environmental samples
  • the purpose of the said gradient dilution is to dilute to the degree of dilution at which the target functional microorganisms disappear, and the highest gradient of dilution can be adjusted according to the total amount of microorganisms in the sample.
  • the gradient dilution process is not limited to 10-fold dilution, and the dilution factor can be adjusted as needed.
  • the gradient dilution is: take 1g or 1ml of environmental sample into a 10ml centrifuge tube, add 9ml of culture medium, which is defined as a 10 -1 dilution sample; then take 1ml of the mixed solution from the 10 -1 dilution to the second 10ml centrifuge tube, add 9ml culture medium, defined as 10-2 dilution sample; then take 1ml mixture from 10-2 dilution to the third 10ml centrifuge tube, add 9ml culture medium, defined as 10-3 dilution sample ; Repeat the similar dilution operation until 10 -8 dilution.
  • the samples with the most degraded substrates and the highest active several dilutions are selected to form the extinct dilution.
  • the culture group is to select the samples with the most degraded substrates and the highest active 3-4 dilutions to form the extinct dilution. Cultivation group.
  • the species identification in step e is to use colony PCR to amplify the 16S rRNA gene for preliminary species identification to verify whether the target species has been isolated.
  • the culture medium is further designed according to the species information of the species. Attempt to separate, but also further scribing purification and identification.
  • the present invention can enrich microbial communities with different functional activity characteristics from environmental samples, especially in complex environmental systems such as soil and sediments, through gradient dilution, and perform high-throughput on these communities. Sequencing, analysis of its structural composition. According to the compositional characteristics of these communities, a separation medium is designed for separation and screening of different types of microorganisms that appear. This method can effectively isolate species with different abundances and different functional characteristics in the environment through the dilution culture process, not only can obtain relatively high abundance species, but also facilitate the separation of relatively low abundance rare species.
  • Figure 1 is a flow chart of the high-throughput analysis and breeding method of functional microorganisms based on the extinct dilution culture group;
  • Figure 2 shows the functional activity characteristics of samples of the extinct dilution culture group of the nitrate-reducing sulfide-oxidizing functional microorganisms in Example 1.
  • A is the reduction of nitrate
  • B is the production of nitrite
  • C is the production of sulfate ;
  • Figure 3 is the community structure of the sample of the extinct dilution culture group of nitrate-reducing sulfide-oxidizing microorganisms in Example 1.
  • Species composition bubble diagram, bubble size indicates the relative abundance of species, S01 and S02 are the species composition of the original sediment sample;
  • Figure 4 shows the functional activity characteristics of samples of the extinct dilution culture group of nitrate-reducing thiosulfate-oxidizing functional microorganisms in Example 2.
  • A is the reduction of nitrate
  • B is the reduction of thiosulfate
  • C is sulfate Production volume
  • FIG. 5 is a bubble diagram of the community structure and species composition of the extinct dilution culture group of the nitrate-reducing thiosulfate-oxidizing functional microorganism in Example 2.
  • FIG. The bubble size indicates the relative abundance of the species.
  • Example 1 The extinct dilution culture group conducts diversity research and high-throughput breeding of nitrate-reducing sulfide-oxidizing microorganisms in river sediments
  • Nitrate-reducing sulfur-oxidizing bacteria-mediated nitrate-reducing sulfur oxidation is an important process that couples the nitrogen cycle and the sulfur cycle, and is the main driving force for the removal of pollutants such as nitrate and sulfide in river sediments.
  • nitrate-reducing sulfur-oxidizing bacteria participate in the nitrate-reducing sulfide oxidation process, it is necessary to first have a full understanding of the diversity and functional characteristics of this type of bacteria in this environment.
  • nitrate-reducing sulfur-oxidizing bacteria in river sediments In the complex sediment environment, there must be many types of nitrate-reducing sulfur-oxidizing bacteria. These nitrate-reducing sulfur-oxidizing bacteria with unknown classification and functional characteristics will hinder our understanding of the process of nitrate-reducing sulfur oxidation in sediments.
  • the extinct dilution culture group was used to conduct high-throughput breeding of nitrate-reducing sulfur-oxidizing bacteria that can use sulfide as a substrate in river sediments to study their diversity and functional characteristics in river sediments.
  • each liter of NS-M medium contains: 2g KNO 3 , 1g Na 2 S ⁇ 9H 2 O, 1g NaHCO 3 , 2g K 2 HPO 4 , 0.1 g MgCl 2 and 1000ml H 2 O, mix well and sterilize for later use.
  • the process of gradient dilution is to take about 1g of river sediments into a 10ml centrifuge tube, add 9ml of NS-M medium, which is defined as a 10 -1 dilution sample; then take 1ml of the mixture from the 10 -1 dilution to the second 10ml Centrifuge tube, add 9ml culture medium, which is defined as 10-2 dilution sample; then take 1ml mixture from 10-2 dilution to the third 10ml centrifuge tube, add 9ml culture medium, which is defined as 10-3 dilution sample; Repeat the operation until a 10 -8 dilution is obtained. A total of 12 repeated gradient dilutions were made and 96 samples were obtained.
  • the change in the substrate was measured. Specifically, ion chromatography is used to determine the concentration of nitrate, nitrite, and sulfate, as well as the biomass, and determine which samples have functional activity through changes in substrates. After gradient dilution, functional differentiation often occurs among the communities of high-dilution samples. Therefore, the highest dilutions with functional activity were selected for community composition analysis. In this example, the highest three dilutions for functional differentiation are 10 -4 , 10 -5 and 10 -6 .
  • These species include Thiobacillus (OTU11), Ciceribacter (OTU11), Rhizobium (OTU3) and Azonexus (OTU5) and Pseudoxanthomonas ( OTU8), etc. (as shown in Figure 3), these may be species with the function of reducing sulfide oxidation by nitrate.
  • Thiobacillus OTU11
  • Ciceribacter OTU11
  • Rhizobium OTU3
  • Azonexus OTU5
  • Pseudoxanthomonas OTU8
  • the significantly enriched species in the extinct dilution culture group were further separated and purified, and representative strains of four dominant genera of Thiobacillus, Ciceribacter, Azonexus and Pseudoxanthomonas, as well as representative strains of non-dominant genera such as Shinella, Ferrovibrio, Hydrogenophaga and Paracoccus were successfully obtained.
  • Representative strains of four dominant genera of Thiobacillus, Ciceribacter, Azonexus and Pseudoxanthomonas as well as representative strains of non-dominant genera such as Shinella, Ferrovibrio, Hydrogenophaga and Paracoccus were successfully obtained.
  • To verify their nitrate reduction sulfide oxidation function although only Paracoccus and Thiobacillus strains can perform nitrate reduction sulfide oxidation, strains of other genera, including Azonexus, Ciceribacter, Ferrovibrio, Hydrogenophaga, Pseudoxanthomonas and Shin
  • Thiobacillus strains have direct sulfide oxidation ability, other strains may indirectly participate in the sulfide oxidation process through metabolic interaction with Thiobacillus.
  • Ciceribacter, Ferrovibrio, Hydrogenophaga and Thiobacillus strains and 16S rRNA gene sequences are relatively low similar to the model strains of related species in the NCBI database, which may be a new species classification. Therefore, the extinct dilution culture group can not only obtain strains with direct functions, but also microorganisms that interact with each other.
  • Thiobacillus and Pseudoxanthomonas are still enriched at 10 -6 dilutions, indicating that they have the highest abundance in the original sediments.
  • Ciceribacter, Rhizobium and Azonexus were enriched at lower dilutions of 10 -4 and 10 -5 , indicating that their abundance in the original sediments was low.
  • the relative abundance of Thiobacillus is about 1.27%, while that of Rhizobium and Azonexus is 0.2-0.3%, and that of Ciceribacter is less than 0.1%.
  • These low-abundance species such as Rhizobium, Azonexus, and Ciceribacter are easily undetectable due to sampling effects in the high-throughput sequencing process. Therefore, the extinct dilution culture group can enrich the low abundance species in the environment, which is further conducive to separation and culture.
  • Example 2 Extinct dilution culture group conducts diversity research and high-throughput breeding of nitrate-reducing thiosulfate-oxidizing microorganisms in sediments
  • thiosulfate is an important intermediate product in the sulfide oxidation process and sulfur cycle.
  • Many nitrate-reducing sulfur-oxidizing bacteria can oxidize thiosulfate, and their thiosulfate oxidation is an important part of the nitrate-reducing sulfur oxidation process in sediments.
  • Diversity of nitrate-reducing sulfur-oxidizing bacteria with thiosulfate oxidation function is also the basis for exploring the process of nitrate-reducing sulfur oxidation in sediments.
  • the source of the sediment in this example is the same as that of Example 1, and the operation process of the extinct dilution culture group is also the same as that of Example 1.
  • the components of the medium are: 2g KNO 3 , 2g NaS 2 O 3 ⁇ 5H 2 O, 1g NaHCO 3 , 2g K 2 HPO 4 , 0.1g MgCl 2 and 1000ml H 2 O.
  • NTS-M medium 2g KNO 3 , 2g NaS 2 O 3 ⁇ 5H 2 O, 1g NaHCO 3 , 2g K 2 HPO 4 , 0.1g MgCl 2 and 1000ml H 2 O.
  • Nitrate consumption rate, thiosulfate consumption rate and sulfate production rate are used to characterize functional activity characteristics.
  • non-dominant communities including Bacillus, Bradyrhizobium, Brevibacterium, Castellaniella, Chelatococcus, Ciceribacter, Ferrovibrio, Mesorhizobium, Ochrobactrum, Paracoccus, Pseudolabrys, Pseudoxanthomonas, Pusillimonas, Ralstonia, Shinotropas, and corresponding Stenotrop strains have been obtained.
  • the nitrate reduction thiosulfate oxidation capacity of the isolated strains was determined, and it was found that all the strains have the nitrate reduction thiosulfate oxidation function (as shown in Table 1).
  • the gene sequence patterns of strains in the 16S BI rRNA database of bacterial strains including Afipia, Aminobacter, Bradyrhizobium, Brevibacterium, Bosea, Castellaniella, Chelatococcus, Ferrovibrio, Hydrogenophaga, Mesorhizobium, Ochrobactrum, Pseudolabrys, Pusillimonas, and Thiobacillus are similar to those in the 16S BI rRNA database They are all relatively low, and they may all be new species classifications.
  • (+) means positive, (-) means negative, ND means not detected.

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Abstract

提供一种基于绝迹稀释培养组的功能微生物高通量分析和选育方法；从环境样品中，特别是如土壤和沉积物等复杂的环境体系中，经过梯度稀释，在不同的稀释度下富集出不同功能活性特征的微生物群落；对这些群落进行高通量测序，分析其结构组成；再根据这些群落的组成特征，针对出现的不同种类微生物，设计分离培养基进行分离筛选，该方法不仅能获得相对丰度高的物种，也能利于分离相对丰度较低的稀有物种。

Description

一种基于绝迹稀释培养组的功能微生物高通量分析和选育方法 技术领域：
本发明属于微生物技术领域，具体涉及一种基于绝迹稀释培养组的功能微生物高通量分析和选育方法。
背景技术：
高通量测序技术和宏基因组学的出现和发展，使我们认识到目前已经分离培养的微生物仅仅是一小部分，环境中超过99％的微生物都仍然未实现纯培养。这些未培养的微生物虽然已经被发现，但是高通量测序获得的信息非常有限，我们很难通过核酸序列信息获知相应物种的功能和表型信息，以及它在环境中的生态作用。此外，生态系统中存在着许多低丰度的稀有物种，这些稀有物种是多样性的重要组成部分。在条件合适时，这些物种可能迅速响应，对推动元素循环和物质转化以及维持生态系统的稳定性发挥着重要的作用。但是，由于高通量测序技术自带的多个步骤的抽样过程，具有一定的检测下限，很难检测到这些稀有物种的存在，更难于研究它们的功能。因此，基于目前高通量测序等技术手段很难对环境中微生物的多样性和功能有全面的认识。大量功能未知的未培养微生物的培养难和低丰度稀有物种的测定难是限制微生物生态学发展的瓶颈。
近年来，培养组学概念的兴起为未培养微生物的挖掘和功能研究提供了新思路。培养组学是通过多种培养条件对目标微生物进行培养，并利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和16S rRNA基因测序技术对它们进行高通量鉴定的一种培养技术。通过培养组，许多研究在人体微生物群，特别是肠道微生物群中分离培养了 大量的新型微生物，更新了我们对人体微生物组的认识。将培养组运用于微生物生态学研究中，将与高通量测序技术等非培养技术形成有效的互补，更加准确的阐明微生物生态学中的问题。培养组技术的特征在于对微生物的高通量鉴定上，而对于环境中特殊类群的微生物仍然需要结合不同的培养方法对不同的微生物种类进行分离。然而，目前对于环境中目标功能微生物类群的选育，仍然缺乏较好的方法。尤其是环境中相对丰度较低的功能物种的选育。
发明内容：
本发明的目的是提供一种基于绝迹稀释培养组的功能微生物高通量分析和选育方法，其能有效分离环境中不同丰度和不同功能特征的物种。
本发明的基于绝迹稀释培养组的功能微生物高通量分析和选育方法，包括以下步骤：
a、利用含底物的选择性培养基对环境样品进行多个系列的梯度稀释；
b、将梯度稀释的样品进行培养，并测定选择性培养基中底物的浓度变化情况；
c、选择降解底物最多的、活性的最高的若干个稀释度的样品组成绝迹稀释培养组，然后收集菌体，按照统一的生物量接种到新鲜的含底物的选择性培养基上进行培养，再测定底物的变化情况，利用底物的变化率反映绝迹稀释培养组不同样品的潜在活性；
d、收集步骤c测定潜在活性后的样品中的菌体，提取总DNA，并扩增16S rRNA基因V4-V5区进行高通量测序，利用生物信息学方法分析绝迹稀释培养组样品微生物群落的物种组成，及其与潜在活性特征的关系；
e、根据绝迹稀释培养组样品的微生物群落组成特征，选取样品先用固体富集培养基对目标功能物种进行单菌落分离筛选，纯化，并进行物种鉴定和功能鉴定，验证是否分离到目标功能物种。
所述的含底物的选择性培养基以及测定选择性培养基中底物的浓度变化情况，是根据筛选目的微生物的功能来决定，例如可以以Na 2S作为底物，来筛选硝酸盐还原硫氧化细菌，硝酸盐还原硫氧化细菌介导的硝酸盐还原硫氧化作用是偶联氮循环和硫循环重要的过程，可以通过离子色谱法测定硝酸盐、亚硝酸盐和硫酸盐的浓度以及生物量，通过底物的变化确定哪些样品具有功能活性。也可以以NaS 2O 3作为底物，来筛选氧化硫代硫酸盐的细菌，硫代硫酸盐是硫化物氧化过程和硫循环的一个重要的中间产物。
所述的环境样品可以是土壤、沉积物、水样等环境样品
优选，所述的梯度稀释的目的是稀释至目标功能微生物绝迹的稀释度，根据样品中总的微生物量，最高梯度的稀释度可以调整。梯度稀释过程不限于10倍稀释，稀释倍数可以根据需要进行调整。
优选，所述的梯度稀释是：取1g或者1ml环境样品到10ml离心管中，加入9ml培养基，定义为10 -1稀释度样品；再从10 -1稀释度取1ml混合液到第二个10ml离心管，加入9ml培养基，定义为10 -2稀释度样品；再从10 -2稀释度取1ml混合液到第三个10ml离心管，加入9ml培养基，定义为10 -3稀释度样品；重复类似的稀释操作，直到10 -8稀释度。
优选，所述的选择降解底物最多的、活性的最高的若干个稀释度的样品组成绝迹稀 释培养组是选择降解底物最多的、活性的最高的3-4个稀释度的样品组成绝迹稀释培养组。
优选，还可以根据绝迹稀释培养组中各个物种分别达到的最高稀释度，分别根据公式“物种个数/每克干沉积物＝(最高位数稀释度稀释倍数/沉积物干重所占质量分数)”估算这些物种在原环境样品中的绝对丰度。
优选，所述的步骤e的物种鉴定是利用菌落PCR扩增16S rRNA基因进行初步物种鉴定，验证是否分离到目标物种，对于群落中无法获得的一些物种，根据物种的种属信息设计培养基进一步尝试分离，同时也进一步划线纯化和鉴定。
本发明能从环境样品中，特别是如土壤和沉积物等复杂的环境体系中，经过梯度稀释，在不同的稀释度下富集出不同功能活性特征的微生物群落；对这些群落进行高通量测序，分析其结构组成。再根据这些群落的组成特征，针对出现的不同种类微生物，设计分离培养基进行分离筛选。该方法通过稀释培养过程，能有效分离环境中不同丰度和不同功能特征的物种，不仅能获得相对丰度高的物种，也能利于分离相对丰度较低的稀有物种。
附图说明：
图1是基于绝迹稀释培养组的功能微生物高通量分析和选育方法的操作流程图；
图2是实施例1中硝酸盐还原硫化物氧化功能微生物的绝迹稀释培养组样品的功能活性特征，A为硝酸盐的减少量，B为亚硝酸盐的产生量，C为硫酸盐的生产量；
图3是实施例1中硝酸盐还原硫化物氧化功能微生物的绝迹稀释培养组样品的群落结构 物种组成气泡图，气泡大小表示物种的相对丰度，S01和S02为原始沉积物样品物种组成；
图4是实施例2中硝酸盐还原硫代硫酸盐氧化功能微生物的绝迹稀释培养组样品的功能活性特征，A为硝酸盐的减少量，B为硫代硫酸盐的减少量，C为硫酸盐的生产量；
图5是实施例2中硝酸盐还原硫代硫酸盐氧化功能微生物的绝迹稀释培养组样品的群落结构物种组成气泡图。气泡大小表示物种的相对丰度。
具体实施方式：
下面结合附图，进一步通过实施例对本发明的基于绝迹稀释培养组对特定功能微生物进行高通量分析和选育的方法进行详细说明。本发明不限于以下实施例的描述。
实施例1：绝迹稀释培养组对河道沉积物中硝酸盐还原硫化物氧化功能微生物进行多样性研究和高通量选育
硝酸盐还原硫氧化细菌介导的硝酸盐还原硫氧化作用是偶联氮循环和硫循环重要的过程，同时是河道沉积物中硝酸盐和硫化物等污染物去除的主要驱动力。硝酸盐还原硫氧化细菌参与硝酸盐还原硫化物氧化过程的阐明，需要首先对该类细菌在该环境中的多样性和功能特征有充分的认识。但是，对于河道沉积物中的硝酸盐还原硫氧化细菌的报道很少。而复杂的沉积物环境必然存在非常多种类的硝酸盐还原硫氧化细菌，这些分类和功能特征未知的硝酸盐还原硫氧化细菌将阻碍我们对沉积物中硝酸盐还原硫氧化过程的认识。
本实施例具体通过绝迹稀释培养组对河道沉积物中能利用硫化物做底物的硝酸盐 还原硫氧化细菌进行高通量选育，以研究其在河道沉积物中的多样性和功能特征。
首先，对河道沉积物进行采样和理化参数的测定，通过测定包括硝酸盐、硫化物和二价铁等浓度的测定表征污染的情况。绝迹稀释培养组的操作流程如图1所示。分别取1g的河道沉积物通过NS-M培养基进行梯度稀释，每升NS-M培养基包含：2g KNO 3、1g Na 2S·9H 2O、1g NaHCO 3、2g K 2HPO 4、0.1g MgCl 2和1000ml H 2O，混合均匀，灭菌备用。梯度稀释的过程是取约1g河道沉积物到10ml离心管中，加入9ml NS-M培养基，定义为10 -1稀释度样品；再从10 -1稀释度取1ml混合液到第二个10ml离心管，加入9ml培养基，定义为10 -2稀释度样品；再从10 -2稀释度取1ml混合液到第三个10ml离心管，加入9ml培养基，定义为10 -3稀释度样品；重复操作，直到获得10 -8稀释度。共做了12次重复的梯度稀释，获得的96个样品。将样品放置于厌氧培养箱30℃培养一个月后，测定底物变化情况。具体是用离子色谱法测定硝酸盐、亚硝酸盐和硫酸盐的浓度以及生物量，通过底物的变化确定哪些样品具有功能活性。经过梯度稀释，高稀释度样品的群落之间往往出现功能分化。因此，选取具有功能活性的最高几个稀释度进行群落组成分析。本实施例中，出现功能分化的最高三个稀释度为10 -4、10 -5和10 -6。分别对这三个稀释度样品的菌体进行收集，按照一致的生物量(最终浓度总蛋白量约1μg/ml)分别接种到新鲜的NS-M培养基的中，放置于厌氧培养箱中30℃培养，分别在0天、3天、5天、7天和9天取样测定底物变化情况，以底物的浓度变化率来表示硝酸盐还原和硫化物氧化活性的大小。此外，分别提取这些样品的总DNA，通用高通量测序引物515F：5’-GTG CCA GCM GCC GCG GTA A-3’(如SEQ ID NO.1所示)和 806R：5’-GGA CTA CHV GGG TWT CTA AT-3’(如SEQ ID NO.2所示)，对16S rRNA基因的V5区进行PCR扩增和高通量测序，通过生物信息学方法分析这些样品的群落组成特征及其与功能活性的关系。最后根据不同样品的物种组成，选择样品利用多种琼脂糖固体培养基对关键微生物菌种进行平板单菌落分离筛选。获得的单菌落通过16S rRNA基因通用引物27F：5’-AGA GTT TGA TCM TGG CTC AG-3’(如SEQ ID NO.3所示)和1492R：5’-TAC GGY TAC CTT GTT ACG ACT T-3’(如SEQ ID NO.4所示)进行PCR扩增和测序，序列拼接后在NCBI数据库进行比对，初步鉴定其分类特征。并测定菌株的硝酸盐还原硫氧化功能活性特征。
结果表明，10 -4、10 -5和10 -6稀释度下的不同系列的样品具有不同的功能活性特征，其中10 -4稀释度的所有样品都具有较高的功能活性，10 -5比10 -4较低，10 -6稀释度下只有几个样品具有显著的功能活性，而且活性较低(如图2所示)。对这些样品的细菌群落组成进行分析，发现没有明显硝酸盐还原硫化物氧化活性的样品，菌群依然非常复杂，没有产生明显的富集效果(如图3所示)。而在具有明显功能活性的样品中，几个物种的不同组合构成了不同样品细菌群落的主体，这些物种包括Thiobacillus(OTU11)、Ciceribacter(OTU11)、Rhizobium(OTU3)和Azonexus(OTU5)与Pseudoxanthomonas(OTU8)等(如图3所示)，这些都可能是具有硝酸盐还原硫化物氧化功能的物种。而这些物种中，只有Thiobacillus被报道过具有硝酸盐还原硫化物氧化功能。
进一步对绝迹稀释培养组中显著富集的物种进行分离纯化，成功获得了Thiobacillus、Ciceribacter、Azonexus和Pseudoxanthomonas四个优势属的代表菌株，以 及Shinella、Ferrovibrio、Hydrogenophaga和Paracoccus等非优势属的代表菌株。对它们进行硝酸盐还原硫化物氧化功能验证，虽然只有Paracoccus和Thiobacillus的菌株能进行硝酸盐还原硫化物氧化，但是其他属，包括Azonexus、Ciceribacter、Ferrovibrio、Hydrogenophaga、Pseudoxanthomonas和Shinella的菌株都具有硝酸盐还原硫代硫酸盐氧化功能。虽然Thiobacillus的菌株有直接的硫化物氧化能力，但是其他的菌株可能通过与Thiobacillus进行代谢互作，间接的参与到硫化物的氧化过程中。此外，Ciceribacter、Ferrovibrio、Hydrogenophaga和Thiobacillus的菌株与16S rRNA基因序列与NCBI数据库中近缘物种模式菌株的相似度都比较低，可能是新的物种分类。因此，绝迹稀释培养组不仅能获得具有直接功能的菌株，还能获得存在互作共生的微生物。
根据这些物种在不同稀释度出现的频数，Thiobacillus和Pseudoxanthomonas菌属在10 -6稀释度下依然被富集起来，说明它们在原沉积物中的丰度最高。而Ciceribacter、Rhizobium和Azonexus等在较低的10 -4和10 -5稀释度下富集，说明它们在原沉积物中的丰度较低。参考最大可能数法(MPN)，用公式：物种个数/每克干沉积物＝(最高位数稀释度稀释倍数/沉积物干重所占质量分数)对这些物种在原沉积物中的绝对丰度进行估算，Thiobacillus和Pseudoxanthomonas的绝对丰度约为10 7个/克干泥，Rhizobium、Azonexus和Ciceribacter的绝对丰度约为10 6个/克干泥。这些结果与原沉积物中总细菌16S rRNA基因高通量测序结果一致，Thiobacillus的相对丰度约为1.27％，而Rhizobium和Azonexus为0.2-0.3％，Ciceribacter则低于0.1％。Rhizobium、Azonexus和Ciceribacter等这些低丰度物种很容易由于高通量测序过程中的抽样效应而无法测到。因此，绝迹稀 释培养组能将环境中低丰度的物种富集起来，而进一步的利于分离培养。
实施例2：绝迹稀释培养组对沉积物中硝酸盐还原硫代硫酸盐氧化功能微生物进行多样性研究和高通量选育
承接实施例1，硫代硫酸盐是硫化物氧化过程和硫循环的一个重要的中间产物。许多硝酸盐还原硫氧化细菌都能氧化硫代硫酸盐，它们进行的硫代硫酸盐氧化作用是沉积物中硝酸盐还原硫氧化过程的重要组成部分。对具有硫代硫酸盐氧化功能的硝酸盐还原硫氧化细菌进行多样性研究也是探索沉积物硝酸盐还原硫氧化过程的基础。
本实施例的沉积物来源与实施例1一致，绝迹稀释培养组的操作流程也与实施例1一致。其中培养基(NTS-M培养基)的成分是：2g KNO 3、2g NaS 2O 3·5H 2O、1g NaHCO 3、2g K 2HPO 4和0.1g MgCl 2和1000ml H 2O。后续的培养、功能活性测定和细菌群落组成分析与实施例1一致。以硝酸盐消耗速率，硫代硫酸盐消耗速率和硫酸盐产生速率表征功能活性特点。
结果发现，10 -7和10 -8稀释度的样品出现大量的功能绝迹，而且10 -5到10 -8的四个稀释度的样品群落的功能活性差异分化较大，群落的功能活性沿着稀释度呈线性下降趋势(图4)。对这几个稀释度的样品进行细菌群落分析，与实施例1一致的是，明显具有硝酸盐还原硫代硫酸盐氧化活性的样品，微生物的群落组成发生了简化，说明产生了富集效果。由Achromobacter、Afipia、Aminobacter、Aridibacter、Azonexus、Bosea、Comamonas、Hydrogenophaga、Micromonospora、Pseudomonas、Rhizobium、Rhodanobacter、Sphingopyxis、Thermomonas和Thiobacillus等属的不同组合构成了多种 多样的群落(图5)。这些属很可能是具有硫代硫酸盐氧化功能的硝酸盐还原硫氧化细菌。其中Achromobacter、Bosea、Pseudomonas、Rhodanobacter和Thiobacillus都已经被报道能进行硝酸盐还原硫代硫酸盐氧化作用。而没有活性的样品，微生物群落多样性依然很高，结构组成复杂。
通过固体培养基对这些物种进行分离纯化，成功获得了包括Achromobacter、Afipia、Aminobacter、Azonexus、Bosea、Comamonas、Hydrogenophaga、Micromonospora、Pseudomonas、Rhodanobacter、Sphingopyxis、Thermomonas和Thiobacillus等优势属对应的菌株。此外，还获得了包括Bacillus、Bradyrhizobium、Brevibacterium、Castellaniella、Chelatococcus、Ciceribacter、Ferrovibrio、Mesorhizobium、Ochrobactrum、Paracoccus、Pseudolabrys、Pseudoxanthomonas、Pusillimonas、Ralstonia、Shinella和Stenotrophomonas等在群落中非优势属对应的菌株。对分离获得的菌株进行硝酸盐还原硫代硫酸盐氧化能力测定，发现所有的菌株都具有硝酸盐还原硫代硫酸盐氧化功能(如表1所示)。其中，包括Afipia、Aminobacter、Bradyrhizobium、Brevibacterium、Bosea、Castellaniella、Chelatococcus、Ferrovibrio、Hydrogenophaga、Mesorhizobium、Ochrobactrum、Pseudolabrys、Pusillimonas和Thiobacillus等菌属的菌株的16S rRNA基因序列与NCBI数据库中的模式菌株的相似度都比较低，它们都可能是新的物种分类。因此，通过绝迹稀释培养组，我们获得了大量以往仍未报道过的具有硫代硫酸盐氧化功能的物种，其中发现了多个可能的新物种。目前已报道具有硝酸盐还原硫氧化功能物种约有50个菌属，而能进行硫代硫酸盐氧化作用的也仅有约30个属。通过本案例，我们把硝酸盐还原硫 氧化细菌扩展到了约70个菌属，而进行硫代硫酸盐氧化功能的物种扩展到了约50个，大大加深了我们对这类细菌多样性的认识。
根据这些物种在不同稀释度出现的频数，用公式：物种个数/每克干沉积物＝(最高位数稀释度稀释倍数/沉积物干重所占质量分数)对它们在原沉积物中的绝对丰度进行估算。如表1所示，估算的结果多数与高通量测序的相对丰度结果一致，相对丰度低的或者无法检测的，估算得到的绝对丰度也较低。表明多数的这些物种在沉积物中的丰度很低，高通量测序技术带有的随机性和抽样效应往往会难以测到这些物种的存在。然而，通过绝迹稀释培养组，我们成功将这些物种富集起来，并通过分离纯化获得了纯培养。因此，通过绝迹稀释培养组，我们发现了沉积物中这些物种的多样性分布规律，大多数的物种丰度很低，它们可能在合适的条件下进行繁殖，发挥它们的生态功能。
表1通过绝迹稀释培养组分离获得的菌株及其功能和在原沉积物中的相对丰度
Figure PCTCN2020141959-appb-000001
Figure PCTCN2020141959-appb-000002
(+)表示阳性，(-)表示阴性，ND表示未检测到。

Claims (7)

  1. 一种基于绝迹稀释培养组的功能微生物高通量分析和选育方法，其特征在于，包括以下步骤：
    a、利用含底物的选择性培养基对环境样品进行多个系列的梯度稀释；
    b、将梯度稀释的样品进行培养，并测定选择性培养基中底物的浓度变化情况；
    c、选择降解底物最多的、活性的最高的若干个稀释度的样品组成绝迹稀释培养组，然后收集菌体，按照统一的生物量接种到新鲜的含底物的选择性培养基上进行培养，再测定底物的变化情况，利用底物的变化率反映绝迹稀释培养组不同样品的潜在活性；
    d、收集步骤c测定潜在活性后的样品中的菌体，提取总DNA，并扩增16S rRNA基因V4-V5区进行高通量测序，利用生物信息学方法分析绝迹稀释培养组样品微生物群落的物种组成，及其与潜在活性特征的关系；
    e、根据绝迹稀释培养组样品的微生物群落组成特征，选取样品先用固体富集培养基对目标功能物种进行单菌落分离筛选，纯化，并进行物种鉴定和功能鉴定，验证是否分离到目标功能物种。
  2. 根据权利要求1所述的基于绝迹稀释培养组的功能微生物高通量分析和选育方法，其特征在于，所述的含底物的选择性培养基中的底物是Na 2S，来筛选硝酸盐还原硫氧化细菌；或以NaS 2O 3作为底物，来筛选氧化硫代硫酸盐的细菌。
  3. 根据权利要求1所述的基于绝迹稀释培养组的功能微生物高通量分析和选育方法，其特征在于，所述的环境样品是土壤样品、沉积物样品或水样品。
  4. 根据权利要求1所述的基于绝迹稀释培养组的功能微生物高通量分析和选育方法，其特 征在于，所述的梯度稀释是：取1g或者1ml环境样品到10ml离心管中，加入9ml培养基，定义为10 -1稀释度样品；再从10 -1稀释度取1ml混合液到第二个10ml离心管，加入9ml培养基，定义为10 -2稀释度样品；再从10 -2稀释度取1ml混合液到第三个10ml离心管，加入9ml培养基，定义为10 -3稀释度样品；重复类似的稀释操作，直到10 -8稀释度。
  5. 根据权利要求1所述的基于绝迹稀释培养组的功能微生物高通量分析和选育方法，其特征在于，所述的选择降解底物最多的、活性的最高的若干个稀释度的样品组成绝迹稀释培养组是选择降解底物最多的、活性的最高的3-4个稀释度的样品组成绝迹稀释培养组。
  6. 根据权利要求1所述的基于绝迹稀释培养组的功能微生物高通量分析和选育方法，其特征在于，还可以根据绝迹稀释培养组中各个物种分别达到的最高稀释度，分别根据公式“物种个数/每克干沉积物＝(最高位数稀释度稀释倍数/沉积物干重所占质量分数)”估算这些物种在原环境样品中的绝对丰度。
  7. 根据权利要求1、2、3、4、5或6所述的基于绝迹稀释培养组的功能微生物高通量分析和选育方法，其特征在于，所述的步骤e的物种鉴定是利用菌落PCR扩增16S rRNA基因进行初步物种鉴定，验证是否分离到目标物种，对于群落中无法获得的一些物种，根据物种的种属信息设计培养基进一步尝试分离，同时也进一步划线纯化和鉴定。
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