Sulfate reducing bacteria[1](Sulfate Reducing Bacteria, SRB for short) is a unique prokaryotic physiological group. It is a strict anaerobic bacteria with various morphological characteristics that can reduce sulfate by dissimilating sulfate as an electronic receptor of organic matter.SRB is widely distributed on the earth and exerts many potential through various interactions, especially in the anoxic aquatic and terrestrial environments caused by microbial metabolism, such as soil, seawater, river water, underground pipelines, oil and gas wells, flooded paddy soil, river and lake sediments, marsh mud and other anaerobic habitats rich in organic matter and sulfate and some extreme environments.
As early as 1924, BENGOUGH and MAY believed that the HtwoS plays an important role in the corrosion of iron components buried underground. In 1934, Dutch scholars Kuer and Vilute proposed that SRBMetal corrosionMechanism of action;Later, Bunker (1939), HEDELAI (1940), Stark and Witt (1945) also confirmed that the main bacteria for corrosion wereIron bacterium(Aerobic) and SRB（anaerobic）The corrosion of steel in soil is mainly the latter.
According to factors such as the growth and reproduction conditions of sulfate reducing bacteria, the mechanism of corrosion activity and the target of action, the prevention and control of SRB corrosion can be divided into physical methods, chemical methodscathodic protection Methods, microbial protection methods andAnticorrosive materialsProtection methods, etc.
However, some of the above methods are either less efficient or more expensive.And like some chemical methods（bactericide）It also brings new burdens to environmental governance.With the increasing awareness of environmental protection, it is particularly important to develop new and efficient environmental protection prevention methods. Preventing SRB corrosion has become a common concern of corrosion science and microbiology.Some anti-corrosion experts believe that from the perspective of environment, it is necessary to find new methods for the prevention and treatment of SRB from microbiology itself.
Growth environment
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Sulfate reducing bacteria (SRB) are widely distributed on the earth and play many potential roles through various interactions, especially in the anoxic aquatic and terrestrial environment caused by microbial metabolism, such as soil, seawater, river and underground pipelines, oil and gas wells, flooded paddy soil, river and lake sedimentsAnaerobic habitats and some extreme environments rich in organic matter and sulfate, such as marsh mud.
SRB is widely distributed in anaerobic environment and water environment, and can be detected by ferrous sulfide precipitation reaction.Marine and sediment are typical habitats of SRB, and there are high sulfate concentrations in these environments.SRB can be detected in the polluted environment, such as rotten food and sewage treatment plant emissions. SRB can also be detected from rice fields, rumen, termite intestines, human and livestock feces and oilfield water[2]。
Classification
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According to incomplete statistics, there are 12 genera and more than 40 species of SRB, and the taxonomic research of SRB is relatively slow.The known SRBs can be divided into two major subcategories in physiology.
asdesulphurizationBacteria, Desulfococcus, DesulfurizationSarcinaAnd desulfurizing bacteria, which can oxidize fatty acids and reduce sulfate to sulfur.With the development of research, some new species have been named.
Cultivation conditions
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Although theoretically, SRB is strictAnaerobic bacteriaHowever, with the deepening of research, existing research results show that SRB is not strictly absoluteanaerobic, but facultative anaerobic.
But generally speaking, SRB is extremely sensitive to oxygen, so the key to its cultivation and separation is to use strict anaerobic technology to cultivate SRB, which requires not only that the surrounding environment is oxygen free, but also that the culture mediumRedox potentialMust be below - 100mV.
Therefore, some strong reducing agents, such asMercaptoethanol、ascorbic acid、L2Cysteine hydrochlorideThese substances are easy to decompose when heated, so they should be sterilized separately by filtration sterilization.
temperature
(1) Medium temperature type: 30-40 ℃;
⑵ High temperature type: 55-60 ℃.
PH value
They can survive within 5-10 days, and the optimal pH value is between 7-8.
For liquid culture of SRB, first remove the air in the culture medium, and then use high-puritynitrogenThe method of blowing off the air in the culture medium and heating the culture medium, then adding a proper amount of bacterial liquid, and standing at a suitable temperature for culture.It is better to cover the culture medium with a layer of sterilized liquid paraffin.
Dilution shake tube method isDilution inverted plate methodAn alternative form ofagarHeat the test tube of the culture medium to melt the agar and keep it at about 50 ℃. Add the diluted bacterial solution with different gradients to these melted agar test tubes and mix them quickly and fully.After coagulation, pour a layer of sterilized liquid paraffin and solid on the surface of the agar columnparaffin waxTo isolate the medium from air as much as possible.After cultivation,colony of bacteriaIt forms in the middle of the agar column.
The difficulty lies in picking up colonies. First, take out the covered paraffin cap with a sterilization needle, and then use anothercapillaryInsert the agar between the tube wall, blow in sterile oxygen free gas, suck out the agar column, and place itPetri dishFinally, the agar column was cut into thin pieces with a sterile knife for observation and colony transfer.
The disadvantage of this method is that it is difficult to observe and pick up colonies, but this method is still convenient and effective in the absence of professional equipmentAnaerobic microorganismThe method of isolation, purification and culture is low cost.
(2) Dish sandwich method
The essence of the dish sandwich method is to clamp bacteria between the upper and lower layers of culture medium, so as to create a relatively oxygen free environment, so that SRB can grow in the crevice.
The specific method is to use the enriched bacterial solutionSterile operationTechnical dilution to different concentrations.Will containmass fraction 2%agarOfSolid mediumMelt and keep at about 50 ℃. Under sterile conditionsPetri dishPour about 1/3 of the height of the solid medium into the (90mm × 15mm) dish cover. After it has just been condensedDiluentSuck an appropriate amount of it, quickly coat the plate, make the diluent permeate for about 30s, pour the same nutrient solid medium into the middle of the culture dish until it is in the state of overflowing, then quickly cover the dish cover and press down, and finally there shall be no bubbles in the dish.
Remove the excess agar between the inner and outer side walls of the culture dish, and fill it with appropriate amount of meltedparaffin waxMake the gap on the side wall of the petri dish sealed with paraffin, and try not to leave bubbles.After one week of cultivation, addFerrous ionBlack SRB will grow in the flat panel ofcolony of bacteria, onAlcohol lampSide heating makes the solid paraffin melt. Because the solidification time of the upper and lower layers of culture medium is different, it is easy to lift the upper layer of culture medium with tweezers after removing the inner dish, thus exposing the colony of the lower layer of culture medium.When colony picking is needed, it can be cut into pieces and transferred intoLiquid mediumIt can be crushed when needed.
The advantage of this method is that the culture is grown onNutrient agarIn the interlayer, it is very convenient to take bacteria at fixed points when taking colonies. At the same time, this method does not need to create an oxygen free environment, so it saves time and effort, and has all the aerobicanaerobicAdvantages of separation methods.
⑶ Hungate rolling technology
Hungate tube rolling technology is to cultivateAnaerobic bacteriaThe best way.The rolling tube technology was first proposed and applied in 1950 by American microbiologist HengaterumenAnaerobic microorganismOne kind of researchAnaerobic cultureTechnology.After decades of continuous improvement, this technology has gradually improved the Hengate anaerobic technology and gradually developed into a complete set of technologies for studying anaerobic microorganisms.Many laboratories specialized in anaerobic culture at home and abroad use this technology.
Hungate tube rolling technology means thatDilutionThe bacteria liquid containingagarIn the anaerobic test tube of the culture medium, then roll it evenly on the tube roller or ice plate, so that the bacteria containing culture medium is uniformly solidified on the inner wall of the test tube.When the agar is completely solidified around the tube wall, the agar tube can be placed vertically for storage, and a small amount of water can be concentrated at the bottom. After several days of culture, it can be seenanaerobicIn pipeSolid mediumInside and on the surfacecolony of bacteriaappear.It is also very convenient to pick up coloniesAlcohol lampSelf made glass tube for side useInoculum needlePick up colonies in good growth condition and quickly receiveLiquid mediuminEnrichment culture。
The advantage of Hungate rolling tube technology is that the culture medium can form a uniform and transparent thin layer on the inner wall of the anaerobic tube, and the colonies can be buried in the culture medium or grow on the surfaceFlat coating methodCompared with oxygen, the chance of contact is greatly reduced[3]。
Role in geochemical cycle
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The combustion of fossil fuels, volcanic eruptions and microbial decomposition are SOtwoThe main source of.In natural state, SO in atmospheretwo, some are absorbed by green plants;Some combine with water in the atmosphere to form HtwoSOfourAs precipitation falls into soil or water, it is absorbed by plant roots in the form of sulfate and transformed into organic matter such as protein, which is then used by consumers at all levels. After the remains of animals and plants are decomposed by microorganisms, they can release sulfur into soil or atmosphere, thus forming a complete cycle.Microbes play an important role in the sulfur cycle, mainly including desulfurization, sulfuration and anti sulfuration[2]。
