Keywords: environmental science; Environmental engineering; Microbiology; environmental microbiology
1 a brief history of microbiology development
Microbiology is a science that studies microorganisms and their life activities. Microbes have many characteristics, such as various types, tiny volume, large specific surface area, various metabolic types, high metabolic intensity, rapid growth and reproduction, easy variation and many kinds. Widely distributed in air, water, soil, human body, animals and plants, living independently or parasitically. Most microorganisms are beneficial to human beings, animals and plants, especially they are closely related to human life and have great influence on industrial and agricultural production, human living environment and health and hygiene [1].
Humans have used microorganisms for a long time. China has rich experience and a long history in using microorganisms. As early as the 3rd century BC, there was a saying in Lv's Spring and Autumn Annals that "Yi Di made wine and drank it, but it was sweet". In the 6th century A.D., The Book of Qi Yao Min written by Jia Sixie at the end of Wei Dynasty recorded in detail the technology of making koji and making wine, and also recorded that planting leguminous plants can enrich the soil. At that time, although I didn't know the existence of rhizobia and the role of nitrogen fixation, I would use rhizobia to accumulate nitrogen fertilizer and so on.
1676, the pioneer of microbiology, Levin Hooke, observed the individual bacteria for the first time with a self-made single microscope, which opened the door to unlock the mystery of the microbial world and was of epoch-making significance. In the next 200 years, people's research on microorganisms only stayed at the low level of describing some microbial forms, including bacteria, protozoa and fungi, without studying their physiological activity mechanism and their relationship with human practice. So at that time, microorganisms as a discipline had not yet formed.
Microbiology was firmly established at the end of 19 and the beginning of the 20th century. Pasteur in France and Koch in Germany are called the founders of microbiology and bacteriology respectively. Pasteur pointed out that fermentation is the function of microorganisms when studying the problem of wine souring in wine production. Pasteur founded the sterilization method and pasteurization method of culture medium and glassware, and established the aseptic technology of dilution and secondary culture. He made outstanding contributions to the development of biology. Koch's great contribution to culture bacteriology is to invent the technology of solid medium "pure culture of bacteria" and purified mixed culture, and to obtain a single purebred by inoculation on solid medium. This technology completely changed the cultivation of bacteriology, and made this subject shine brilliantly in the last twenty years of 19 century. In the 20th century, microbiology achieved rapid development, and the research focus gradually shifted to biochemistry and molecular biology. Industrial microbiology, fermentation engineering, genetic engineering, cell engineering, enzyme engineering and bioreactor engineering together constitute the high-tech field of modern bioengineering.
With the needs of social and economic development, people continue to strengthen the research on microorganisms and form many branches of microbiology. For example, there are general microbiology, microbial physiology, microbial ecology and microbial genetics; There are virology, bacteriology and mycology; There are also soil microbiology and marine microbiology; Then there are medical microbiology, industrial microbiology, agricultural microbiology, environmental microbiology, petroleum microbiology and so on. It can be seen that the development of microbiology reflects the intersection of disciplines, especially the mutual promotion among related cell biology, biochemistry, genetics and molecular biology, thus promoting the rapid development of the whole life science. At the same time, the development of physics, chemistry, computer technology and material science provides the necessary technical means for the development of microbiology. Therefore, the history of microbiology is brilliant [2~4].
2. Research contents of environmental microbiology.
Environmental microbiology mainly introduces the development of environmental microbiology and the basic knowledge of biology; The main types and characteristics of microorganisms existing in the environment; The physiology and metabolism of microorganisms, the influence of microbial growth and environmental factors on microorganisms; Inheritance and variation of microorganisms, ecology and distribution of microorganisms; The existence and change of microorganisms under various environmental conditions, and the influence of microbial metabolic activities on the environment; The position and function of microorganism in nature and its role in biogeochemical cycle; The role of microorganisms in the environmental field, the molecular mechanism of microbial resistance to pollutants and the metabolic pathway of pollutant degradation.
3. Development of environmental microbiology
With the requirements of human life and the rapid development of industrial and agricultural production, a large number of synthetic pollutants that are difficult to be rapidly degraded and transformed by natural microorganisms enter the natural environment. Seriously threaten the normal survival and development of human beings and other creatures. Among them, polycyclic aromatic hydrocarbons, pesticides and other important organic pollutants ubiquitous in soil environment are considered as dangerous substances because of their carcinogenicity, teratogenicity and mutagenicity. The application of molecular biology technology provides new ideas and methods for pollution control and prevention [5]. At the same time, pollution leads to biological recombination in resources and environment, so it is more and more important to monitor and identify complex mixed microbial communities in the environment in time. Molecular biology technology is gradually replacing some traditional research methods, and is widely used in the monitoring of environmental microorganisms because of its rapid, accurate and sensitive characteristics [6].
3. 1 Various molecular biology techniques related to the study of environmental microorganisms
3. 1. 1 nucleic acid probe detection technology
DNA sequence and fragment length polymorphism are analyzed by using known nucleotide fragments that can be specifically complementary to specific nucleotide sequences as probes. Labeled probes (radioactive or non-radioactive) can be directly used to detect solutions, cell tissues or homologous nucleic acid sequences immobilized on membranes by different methods such as in situ hybridization, Southern blot hybridization, dot blot hybridization and narrow line blot hybridization. Because of the high specificity of nucleic acid molecular hybridization and the high sensitivity of detection methods, nucleic acid molecular hybridization technology is widely used to detect microorganisms in the environment, and to qualitatively and quantitatively analyze their existence, distribution, abundance and adaptability.
Fluorescence in situ hybridization (FISH) is a common molecular ecological method to analyze microbial community structure at the single cell level. At present, FISH method can be used to quickly classify a single cell using a set of specific oligonucleotide probes [7~8]. Flow cytometry (FCM) is another technique that can quickly analyze and classify single cell populations. It is suitable for quickly and frequently monitoring the composition and dynamics of microbial communities. RFLP marker is based on Southern blot hybridization, that is, DNA restriction fragment length polymorphism, which is a widely used DNA molecular marker in biodiversity research. It can be used as a highly sensitive method to detect the changes of microbial population in polluted environment.
3. 1.2 PCR and related technologies
PCR is a technique to amplify nucleic acid sequences in vitro to obtain multiple copies of nucleic acids. According to the different amplification templates, primer sequence sources and reaction conditions, PCR techniques can be divided into the following categories: (1) Reverse transcription PCR technique is a PCR amplification of mRNA after reverse transcription, which can be used to analyze the correlation of mRNA expression states at different growth stages. (2) Competitive PCR is a quantitative PCR. The concentration of target template was determined by adding artificial competitive template with mutation into PCR reaction system and controlling the concentration of competitive template, and the target template was quantitatively studied. Competitive PCR has been used to determine the concentration of dmpB gene encoding catechol-2,3-dioxygenase in sediments polluted by polycyclic aromatic hydrocarbons [9]. (3) The restriction endonuclease digestion analysis technology of amplified rDNA is a modern biological identification technology recently developed in the United States. According to the conservation of prokaryotic rDNA sequences, the amplified rDNA fragments are cut by digestion. Then the diversity of bacteria was analyzed by enzyme digestion map.
3. 1.3 electrophoretic separation and display method
In addition to the well-known silver staining methods of agarose gel electrophoresis, ethidium bromide (EB) staining and polyacrylamide gel electrophoresis (PAGE separation), there are some molecular markers established by special electrophoresis separation techniques. Such as denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis and single strand conformation polymorphism. The double helix structure of DNA is changed by implementing some denaturation conditions, such as adding denaturant with linear gradient or establishing denaturation temperature gradient. ). Because DNA fragments with different sequences have different melting degrees, different structures have great influence on the migration rate of DNA in the gel, which leads to the separation of DNA fragments with different sequences on the gel with high resolution.
3. 1.4 gene recombination technology
The process of separating the gene or DNA fragment of interest from the donor genome by DNA recombination or in vitro amplification, or obtaining the gene by artificial synthesis, and then generating recombinant DNA molecules through a series of cutting, processing, modification and ligation reactions, and then transferring them to appropriate recipient cells to obtain gene expression. This technology can be used to study environmental microorganisms, construct various recombinant bacteria with enhanced biodegradation characteristics, and be used for the treatment and restoration of polluted environment or the fermentation of some wastes to produce natural gas. For example, the gene of microbial decomposition of cellulose and lignin is transferred to mesophilic bacteria, so that fermentation can be carried out at higher temperature and the transformation speed can be improved, and it can be used to produce natural gas by sucrose fermentation [10].
3. 1.5 gene chip technology
Gene chip technology is the most developed branch of biochip technology. Gene chip can be divided into cDNA chip and oligonucleotide chip. There are many methods to prepare cDNA chips, which are of great value in the related research of gene expression. Oligonucleotide chips are mainly used for hybridization sequencing, single nucleotide polymorphism analysis and mutation detection. Gene chip, also known as DNA microarray, refers to a micro-lattice array composed of thousands of nucleic acid molecules fixed on a solid carrier in a small area according to a predetermined position. Under certain conditions, nucleic acid molecules on the carrier can hybridize with nucleic acid fragments with complementary sequences in the sample. If the nucleic acid fragment in the sample is labeled, the hybridization signal can be detected on a special chip reader. CDNA chip consists of thousands of CDNA molecules fixed on solid carriers such as glass, silicon wafer, polypropylene film, nitrocellulose film and nylon film.
3.2 Application of Molecular Biology Technology in Environmental Microbiology Research
3.2. 1 Recombinant bacteria for environmental pollution prevention and control
Pseudomonas. P2 is a formyl phosphate degrading bacterium. Luo Ruxin et al. (1 999) [1] cloned the catechol1,2- dioxygenase gene of chlorobenzene degrading bacterium L/kloc-0, and transformed it into P2 bacteria with expression vector PKT230 to make P2.
Plant rhizosphere is a potential site for pollution degradation. In the rhizosphere of plants, plants provide nutrients that microorganisms can use, so that microorganisms can multiply in large numbers and eventually eliminate pollutants. The expression of enzymes related to detoxification in exogenous microorganisms can also provide specific selection advantages in field application. In this case, trichloroethylene (TCE) in soil is detoxified by wheat rhizosphere microorganisms. D.C.yec et al. (1998)[ 12] After the toluene o- monooxygenase gene of Burkholderia cepacia G4 was transformed into Pseudomonas fluorescens 2-79, Pseudomonas fluorescens grew better than other wheat rhizosphere bacteria.
3.2.2 Intracellular enzymes are better expressed on the cell surface and play a role in biodegradation.
Higher plants can produce corresponding metallothionein (MTs) when heavy metals exist. MTs is a cysteine-rich protein, which can bind heavy metal ions such as cadmium, chromium, mercury and copper. Expression of MTs in E.coli is a promising technology, but the adsorption capacity of MTs to metal ions in cells is limited. One way to solve this difficulty is to express MTs on the cell surface. It is reported that inserting MTs at 153 of LamB sequence [13] confirmed this possibility. The expression of hybrid protein increased the binding force with cadmium by 15~20 times. Because MTs is located on the cell surface, it can still be used for metal adsorption and accumulation even if the cell dies. Organophosphorus is a widely used pesticide in agricultural production, and organophosphorus hydrolase (OPH) isolated from soil microorganisms can effectively degrade these pesticides. However, the cost of OPH purification is high, and the detoxification of intact cells is limited by transporting organophosphorus across the cell membrane barrier. The whole cell expressing 0PH on the cell surface degrades phosphorus! Sulfur and paraoxon are several times faster than cells expressing OPH in cells [14]. The biocatalyst with enzyme activity on the cell surface is obviously more stable and active than the purified biocatalyst.
3.2.2 Application of molecular biology technology in environmental microorganism monitoring
Among a large number of microorganisms in the environment, only 65,438+0% can be cultured and further isolated in Petri dishes by traditional culture methods, and most bacteria need very strict nutritional conditions or are difficult to cultivate [65,438+05] PCR technology and a series of biotechnology derived from it make it possible to detect and study a specific gene in low-content population members or biota in those complex environments.
Fantroussi et al. [16] used PCR(nestedPCR to monitor the types of exogenous genes of 3- chlorobenzene dechlorination bacteria in the experimental ecosystem of unpolluted soil sludge. Selvaratnam et al. [17] amplified the dmpN gene encoding phenol monooxygenase by PCR, and detected phenol-degrading Pseudomonas in the batch reactor of wastewater treatment. PCR technology is also applied to environmental monitoring in combination with other methods. Chandler et al. [18] used MPN/PCR technology to estimate the number of naphthalene peroxidase gene nahAc, alkane monooxygenase gene alkB and dmpB in fuel-contaminated soil. The combination of PCR technology and nucleic acid hybridization technology is used to further detect PCR amplification products and verify that the amplified sequence is the target sequence.
The research difficulty of environmental microorganism monitoring microorganisms in the environment is that it is often impossible to accurately, qualitatively and directly detect many microbial populations that may exist in the environment. The appearance and development of gene chip technology provide efficient and fast technical means for researchers in the field of microbiology, which makes it possible for scientific research to develop rapidly in depth [19]. As molecular markers, some genetically engineered bacteria have also been applied to environmental biotechnology, mainly to monitor the situation of microorganisms put into the environment. Many scholars have cloned luxgene from bacteria and transferred it to special microorganisms with decomposition ability, and bacteria with decomposition ability have specific fluorescent markers. By monitoring the fluorescent bacteria and the amount of fluorescence, we can trace special bacteria and understand their relationship with other strains in the decomposition process [20].
3.2.3 Application of Molecular Biotechnology in Stability and Safety Research of Environmental Genetically Engineered Bacteria
The environmental safety of genetically modified organisms needs long-term systematic research, because the emergence of risks has a long-term lag. The stability of introduced genetic material and whether new genetic material is transferred to other microorganisms can be preliminarily judged by biological phenotype, and further confirmed by DNA sequence analysis and probe hybridization. Regarding biological containment, the purpose is to improve the predictability of the behavior and fate of recombinant microorganisms. Biological containment systems can be passive, such as introducing culture defects, recA- and other mutations into strains; Or an active system, pointing to introducing suicide genes into cells and inducing cell death. As the basic principle of biological containment, the strategy of chemically induced suicide is to link the killing function with the regulation system of biodegradation pathway. Cells survive in the presence of abiotic substances (suicide genes are inhibited), but after abiotic substances are completely degraded, suicide genes are turned on and cells die.
With the development of molecular biology technology and the in-depth study of environmental microorganisms, the application of molecular biology technology in environmental microorganisms is more and more extensive and important. The application of molecular biology technology not only broadens the research scope of environmental microorganisms, but also deepens the research depth. With the decoding of all gene sequences of more and more microorganisms, the distribution and expression of degradation genes in various bacteria will be more deeply understood. The maturity of this technology will certainly have a holistic and systematic understanding of environmental microorganisms, and will certainly make the research more targeted and the application more controllable [2 1].