In chronological order, the development of microbial community structure and diversity analysis technology can be divided into three stages. Before the 1970s, the traditional methods of culture and separation were mainly used to classify, identify and count by comparing morphology, culture characteristics and physiological and biochemical characteristics. The understanding of the community structure and diversity of environmental microorganisms is not comprehensive and selective, and the resolution of the method is low. In 1970s and 1980s, researchers summarized some regular conclusions by analyzing the chemical composition of microorganisms, and established some methods of microbial classification and quantification, namely biomarker method, which made the understanding of the structure and diversity of environmental microbial communities enter a more objective level. In 1980s and 1990s, modern molecular biology technology took DNA as the target, accurately revealed the species and genetic diversity of microorganisms through the methods of rRNA gene sequencing and gene fingerprinting, and gave intuitive information about community structure.
1 traditional culture and separation methods
The traditional culture separation method is the earliest method to understand the microbial community structure and diversity, since the invention of 1880.
It is still widely used today. The traditional culture and separation method is to inoculate a quantitative sample into the culture medium, cultivate it at a certain temperature for a certain period of time, and then calculate the content of growing colonies statistically. By observing their morphological structure under the microscope and observing the physiological and biochemical characteristics in the process of culture and separation, the species classification characteristics are identified. The culture separation method adopts simple nutrient substrate and fixed culture temperature, ignoring the influence of climate change and biological interaction. This deviation of the artificial environment from the original habitat greatly reduces the culturable species (only accounting for 0.1%~10% of the total number of environmental microorganisms [1]). Moreover, this method is cumbersome and time-consuming and cannot be used to monitor the dynamic changes of population structure.
2 Community-level physiological fingerprinting (CLPP)
It is generally believed that enzymes contained in microorganisms are closely related to their abundance or activity. Enzyme molecules are highly specific to catalyzed biochemical reactions, and different enzymes participate in different biochemical reactions. If a microbial community contains a specific enzyme that can catalyze the utilization of a specific substrate, this enzyme-substrate can be used as one of the biomarkers of this community, marking the existence of a certain group. Community-level physiological fingerprint (CLPP) proposed by Garlan and Mills[2] in 199 1 is an enzyme activity analysis method, which reflects the composition of the population by detecting the substrate utilization pattern of microbial samples. Specifically, CLPP analysis is to determine which substrates can be used as energy by detecting the utilization ability of microbial samples to a variety of different single carbon source substrates, thus generating physiological metabolism fingerprints of substrate utilization. BIOLOG redox technology developed by BIOLOG company makes CLPP method fast and convenient. There are two types of BIOLOG microplates in the market: GN and MT, both of which contain 96 wells, each of which
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Microplate dry film contains culture medium and redox dye tetrazole [3]. Among them, BIOLOG's GN microplate contains 95 different carbon sources and a control well without carbon source, while MT microplate only contains culture medium and redox dyes, allowing free detection of different carbon source substrates [3]. The detection method is: add the treated microbial samples into each micropore and incubate at a certain temperature for a certain time (generally 12 h). During incubation, redox dyes are reduced by NADH produced by respiratory pathway, and the rate of color change depends on respiratory rate. Finally, the absorbance of a certain wavelength is detected, and the utilization type and degree of energy carbon are analyzed [4].
BIOLOG method can effectively evaluate the microbial community structure of soil and other environmental flora [3~6]. Its advantage is that the operation is relatively simple and fast, and a small amount of carbon sources can distinguish the differences in carbon utilization methods [5]. However, BIOLOG system can only identify fast-growing microorganisms. In addition, the near-neutral buffer system, high concentration of carbon source and biotoxicity indicator red tetrazole (TTC) in the test tray further increase the error of the test results. Yao's research shows that the BIOLOG system should be improved according to the characteristics of the test object (such as pH, carbon source utilization type, utilization ability, etc.), and better indicators should be studied to replace it.
3 biomarker method
Biomarkers are usually biochemical components of microbial cells, and their total amount is usually positively correlated with the corresponding biomass. Because specific structural markers mark specific types of microorganisms, the composition patterns (species, quantity and relative proportion) of some biomarkers can be used as fingerprints to evaluate the microbial community structure. Because the classification is based on the biochemical components extracted from mixed microbial communities, it potentially includes all species, so it has certain objectivity. It is suitable for qualitative or even semi-quantitative detection of dynamic changes of microbial system. Since 1980s, biomarker methods commonly used to study microbial community structure include quinones and fatty acids (PLFAs and WCFA-FAMEs). In the process of determination, these compounds are extracted and purified from environmental microbial samples with appropriate extractant, and then made into appropriate samples with appropriate solvents for GC or LC detection. Finally, the obtained biomarker spectrum was qualitatively and quantitatively analyzed by statistical method.
3. 1 quinone analysis
Respiratory quinone widely exists in the cell membrane of microorganisms, is a component of cell membrane, and plays an important role in the electron transfer chain [7]. The good linear relationship between the content of quinone and the biomass of soil and activated sludge shows that the content of quinone can be used as a indicator of microbial biomass [8]. There are two main types of respiratory quinones: ubiquinone (UQ) namely coenzyme Q and menaquinone (MK) namely vitamin K[9]. According to the number of isoprene-containing units in side chains and side chains, quinone can be saturated by double bonds on the basis of molecular structure (UQ and MK)
The number of hydrogen atoms is further distinguished. Studies have shown that each microorganism contains a dominant quinone [7], and different microorganisms contain quinones with different types and molecular structures [9]. Therefore, the diversity of quinone can quantitatively characterize the diversity of microorganisms, and the change of quinone spectrum (that is, quinone fingerprint) can characterize the change of community structure.
The parameters [7] of quinone fingerprint used to describe microbial community are: (1) the types of quinones and the number of different quinones; (2) Dominant quinone and its molar fraction; (3) the ratio of total ubiquinone to total methylnaphthoquinone; (4) Diversity and uniformity of quinones; (5) The total amount of quinone, etc. For two different communities, another parameter _ _ (dissimilarity index, D) can be calculated from the data obtained from the above analysis, which can be used to quantitatively compare the structural differences between the two communities.
Quinone fingerprinting method is simple and rapid, and has been widely used in the analysis of various environmental microbial samples (such as soil, activated sludge and other aquatic environmental communities) in recent years.
The accuracy of quinone fingerprint method in activated sludge community analysis was investigated, which proved that this method is a reliable analysis method. However, the quinone fingerprint method also has some limitations and cannot reflect the changes of specific genera or species. 3.2 fatty acid spectrum method (PLFAs, PLFAs, Metz and other methods)
Biochemical components of fat extracted from microbial cells are important biomarkers of biomass, for example, polar lipids (phospholipids) and neutral lipids (diglycerides) can be used as markers of active and inactive biomass respectively [10]. More importantly, the extraction of mixed long-chain fatty acids with different molecular structures of lipid decomposition products implies the type information of microorganisms, and its composition mode can be used as a symbol of population composition. Fatty acid spectrum method is widely used for typing and dynamic monitoring of microbial community structure in soil, compost and water environment [1 1~ 13].
Commonly used fatty acid spectrograms can be divided into two types: phospholipid fatty acid (PLFAs) spectrogram and whole cell fatty acid methyl ester (WCFA- Fames) spectrogram [14]. Both of them are essentially fatty acid methyl esters, and the difference lies in the different sources of fatty acids. Fatty acids extracted by phospholipid fatty acid (PLFAs) spectrometry mainly come from microbial cell membrane phospholipids, that is, living cells, and fatty acids extracted by whole-cell fatty acid methyl ester (PLFAs Fames) spectrometry come from all methylated lipids in environmental microbial samples, that is, all cells (including living cells and dead cells). Therefore, the advantage of phospholipid fatty acid (PLFAs) spectrum is accurate and reliable. The advantages of whole cell fatty acid methyl ester (WCFA- Fames) spectrogram method are simple extraction and less sample consumption. For the analysis of a variety of environmental microbial samples, it is a good choice to pre-screen by PLFAs method and then analyze by PLFAs method.
The analysis of fatty acid spectrum includes two forms: one is fatty acid, and molecules with different structures are separated by GC analyzer; The other is fatty acid methyl ester, the product of fatty acid methylation. Different molecules were separated and identified by GC-MS analyzer.