CN104745519A - Method for screening dissimilatory Fe(III)-reducing bacteria - Google Patents

Method for screening dissimilatory Fe(III)-reducing bacteria Download PDF

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CN104745519A
CN104745519A CN201510183645.8A CN201510183645A CN104745519A CN 104745519 A CN104745519 A CN 104745519A CN 201510183645 A CN201510183645 A CN 201510183645A CN 104745519 A CN104745519 A CN 104745519A
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刘洪艳
王红玉
马帅
殷艳艳
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Tianjin University of Science and Technology
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Tianjin University of Science and Technology
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells

Abstract

The invention discloses a method for screening dissimilatory Fe(III)-reducing bacteria. The method comprises the following steps: (1) carrying out enrichment culture on sediment sludge by way of gradually improving the Fe(III) concentration in a liquid culture medium; and (2) separating and purifying the dissimilatory Fe(III)-reducing bacteria from enriched flora and carrying out gradient-acclimation on heavy metal endurance capacity and salt tolerance. The method for screening dissimilatory Fe(III)-reducing bacteria is simple and feasible and short in screening period. In the screening process, expensive separating equipment such as an anaerobic operation system and the like is required, so that the screening cost is effectively lowered. The obtained pure strain has a relatively strong Fe(III) reducing capacity. In addition, through gradient-acclimation on heavy metal endurance capacity and salt tolerance of the pure strain, a dissimilatory Fe(III)-reducing bacterial strain which has the endurance capacity to Cr (VI) heavy metals and different salt concentration environments can be quickly and simply obtained, thereby providing a germplasm resource for biological repair in the environments such as a heavy metal region, a salt region and the like.

Description

A kind of method of screening dissimilatory iron reduction bacterium
Technical field
The present invention relates to technical field of environmental microorganism, particularly relate to a kind of method of screening dissimilatory iron reduction bacterium.
Background technology
Iron is a kind of variable valency metal, and the biological oxido reduction of iron relates to the biogeochemical cycle of multiple element.The ferrikinetics process of taking as the leading factor with Fe (III) and Fe (II) is one of key factor of regulation and control heavy metal transformation conversion, and the dissimilatory iron reduction process of microbe-mediated can realize organic pollutant oxygenolysis under anaerobic simultaneously.Therefore, microbe-mediated Fe3+ reduction process has vital role in ecotope and biological restoration.
Due to the fast development of modern industry, organic pollutant accumulation progressively in environment, causes many contaminated environment to provide the condition of a kind of anaerobism or anoxic.And under anaerobic environment, Fe (III) becomes the most important electron acceptor(EA) of oxidation operation.Dissimilatory iron reduction bacterium is not the concept on a taxonomy, and it is actually the general name of a quasi-microorganism with Fe (III) restoring function.The microorganism with dissimilatory iron reduction ability is all found (Lee and Newman in bacterium and archeobacteria territory, Microbial iron respiration:impacts on corrosion processes.Applied and EnvironmentalMicrobiology, 2003,62:134-138).Up to now, at 9 doors in bacterium territory, 15 guiding principles, 27 orders, 37 sections, 65 belong to and 2, archeobacteria territory door, 5 guiding principles, 7 orders, 7 sections, the distribution of dissimilatory iron reduction microorganism is found that there is in 10 genus, table 1 is part dissimilatory iron reduction microorganism and source (Lovley thereof in relevant settling, Bioremediation of organicand metal contaminants withdissimilatory metalreduction.Journal of Industrial Microbiology & Biotechnology, 1995, 14:85-93).
Dissimilatory iron reduction bacterium and source thereof in table 1 Parts of deposits
Dissimilatory iron reduction bacterium has comparatively clear superiority in pollution law: (1) transfer heavy metal, except Fe (III), dissimilatory iron reduction bacterium can also utilize other metal as: Mn (IV), U (VI) and Cr (VI) are as electron acceptor(EA), effective reduction harmful heavy metal, reduces toxicity; (2) decomposing organic matter, the content of ferriferous oxide in the ecosystem is very abundant, and in the region, deep-sea that amorphous iron oxide content is abundanter, dissimilatory iron reduction can reach 75% to the contribution of organic substance decomposing; (3) degradable organic pollutant, dissimilatory iron reduction bacterium can utilize many organic pollutants, if benzene and aromatics are as electron donor.
The dissimilatory iron reduction process of microbe-mediated can fully be carried out under anaerobism or amphimicrobian condition, and therefore screening has the dissimilatory iron reduction bacterium of anaerobism character is the common recognition that investigators are formed.But, conventional anaerobic operation systematic position method not only apparatus expensive, and the screening cycle is long, hinders the process of the screening operation of dissimilatory iron reduction bacterium.Therefore, provide a kind of cheapness, practicality and the screening cycle short dissimilatory iron reduction microorganism screening method be necessary.
Summary of the invention
The object of this invention is to provide a kind of method of screening dissimilatory iron reduction bacterium, this screening method has easy, fast and the low feature of cost, by this screening method can rapid screening to dissimilatory iron reduction bacterium, for utilize microorganism dissimilatory iron reduction process administer heavy metal contamination germ plasm resource is provided.
In technical scheme of the present invention, the screening method of dissimilatory iron reduction bacterium comprises the steps:
(1) mode progressively improving Fe (III) concentration in liquid nutrient medium is utilized to carry out enrichment culture to settling mud;
(2) separation and purification dissimilatory iron reduction bacterium from enrichment flora;
Carrying out the method for enrichment culture to settling mud in described step (1) can for the existing any method in this area, and the mode preferably improving Fe (III) concentration in substratum gradually carries out enrichment culture; Described settling mud for inoculating can choose the settling mud under the anaerobic environment commonly used this area, the settling bed mud in preferred various waters.Concrete grammar is as follows: added by mud sample in the liquid nutrient medium containing Fe (III), inflated with nitrogen Anaerobic culturel, in liquid nutrient medium, the starting point concentration of Fe (III) is 5 ~ 10mmol/L, flora stronger for Fe (III) reducing power is progressively forwarded in the higher liquid nutrient medium of Fe (III) concentration in culturing process, the concentration of Fe (III) is improved 5 ~ 10mmol/L, until bring up to 50mmol/L by each switching.The flora that described Fe (III) reducing power is stronger refers to after improving Fe (III) concentration at every turn, through cultivation after a while, can make OD in liquid nutrient medium 600be worth>=0.500 and the flora of Fe (II) concentration>=1mmol/L.Those skilled in the art should understand, the OD of liquid nutrient medium 600value reflects the growth conditions of bacterium, under normal circumstances, works as OD 600when value is between 0.300 ~ 0.600, OD 600value is higher shows that Bacteria cold shock is better, and cell density is larger, is in the comparison that flora under this state or bacterial strain are suitable for carrying out katabolism ability; Work as OD 600be worth lower than 0.300 time, show that the growth conditions of bacterium is poor, lack the tolerance to respective environment.Therefore, this step and follow-up in steps in OD in liquid nutrient medium is all selected to the mensuration of Fe (II) and Cr (VI) concentration 600the flora of value>=0.500 or bacterial strain, for the ease of comparing, the OD of unified rearrange liquids substratum before measuring 600value is to 0.500 ± 0.02.
The method of described step (2) separation and purification dissimilatory iron reduction bacterium from enrichment flora is as follows: adopt plate streak to carry out separation and purification to the flora with obvious Fe3+ reduction ability obtained by step (1).Detailed process is as follows: by centrifugal for the mud suspension after step (1) enrichment culture, after getting the dilution of supernatant bacterium liquid, streak inoculation is on the solid medium containing Fe (III) (concentration is 10 ~ 50mmol/L), the identical solid medium of one deck is toppled over again to ensure bacterium liquid and air exclusion after bacterium liquid is absorbed by solid medium completely, after the solid medium on upper strata solidifies, topple over the capping in addition of one deck whiteruss again, to guarantee the anaerobic culture environment of bacterium colony; By picking list bacterium colony after this three layer flat plate cultivation 24 ~ 72h, be inoculated in the liquid nutrient medium containing Fe (III) (concentration is 10 ~ 50mmol/L), the concentration of Fe in liquid nutrient medium (II) is measured after inflated with nitrogen Anaerobic culturel 24 ~ 72h, choose accumulation Fe (II) bacterial strain that concentration is the highest, again be inoculated in described three layer flat plate and carry out cultivating and being separated, repeat said process, until obtain purifying bacterial strain.
Owing to polluting in comparatively serious water surrounding often containing a large amount of heavy metal ion and higher salinity at trade effluent etc., in order to enable dissimilatory iron reduction bacterium in above-mentioned extreme environment survival and keep stronger dissimilatory iron reduction ability, the screening method of dissimilatory iron reduction bacterium of the present invention also can comprise the gradient domestication step of the follow-up heavy metal that carries out and high salinity tolerance, to obtain, application performance better bacterial strain stronger to adaptive capacity to environment.
Technical solution of the present invention adopts the concentration progressively improving Cr (VI) in substratum to carry out the gradient domestication of dissimilatory iron reduction bacterium heavy metal tolerance.Concrete grammar is as follows: the purifying inoculation that step (2) is obtained in containing Fe (III) (concentration is 10 ~ 50mmol/L) liquid nutrient medium in, inflated with nitrogen Anaerobic culturel.In culturing process, heavy metal tolerance and the stronger bacterial strain of Fe (III) reducing power are progressively forwarded in the higher liquid nutrient medium of Cr (VI) concentration, in described liquid nutrient medium, the maximum concentration of Cr (VI) is no more than 2.5mmol/L, and the concentration of Fe (III) remains unchanged.Described heavy metal tolerance and the stronger bacterial strain of Fe (III) reducing power through cultivation after a while, can make OD in liquid nutrient medium after referring to and improving Cr (VI) concentration at every turn 600be worth>=0.500 and the bacterial strain of Fe (II) concentration>=1mmol/L.
Survive under the environment of higher salt concentrations to enable dissimilatory iron reduction bacterium and keep stronger dissimilatory iron reduction ability, technical solution of the present invention adopts the method progressively improving salt concn in substratum the bacterial strain obtained by described step (2) to be carried out to the gradient domestication of high salinity tolerance, and concrete grammar is as follows: purifying inoculation step (2) obtained is inflated with nitrogen Anaerobic culturel in the liquid nutrient medium containing Fe (III) (concentration is 10 ~ 50mmol/L).In culturing process, salt resistance ability and the stronger bacterial strain of Fe (III) reducing power are progressively forwarded in the higher liquid nutrient medium of NaCl concentration, in described liquid nutrient medium, the maximum concentration of NaCl is no more than 60g/L, and the concentration of Fe (III) remains unchanged.Described salt resistance ability and the stronger bacterial strain of Fe (III) reducing power through cultivation after a while, can make OD in liquid nutrient medium after referring to each raising NaCl concentration 600be worth>=0.500 and the bacterial strain of Fe (II) concentration>=1mmol/L.
The described liquid nutrient medium containing Fe (III) that each step of technical solution of the present invention (comprising the gradient domestication step of described step (1), step (2) and heavy metal and high salinity tolerance) uses is for adding Fe (III), and must the common liq substratum of nutritive substance (comprising carbon source, nitrogenous source, the energy, somatomedin and inorganic salt etc.) containing microorganism growth, optimization citric acid iron liquid nutrient medium; The solid medium containing Fe (III) used in described step (2) for adding Fe (III), and contains the required nutritive substance ordinary solid substratum of microorganism growth, optimization citric acid iron solid medium.The potential of hydrogen of above-mentioned all substratum all remains on: pH6.0 ~ 9.0, and corresponding culture temperature is: 20 ~ 40 DEG C.
For the settling mud taken from the environment of Deng Yan territory, ocean, also be added with NaCl in the substratum that uses in the gradient domestication step of described step (1), step (2) and heavy metal tolerance, the salt concn in physical environment residing for the concentration of NaCl and settling mud is basically identical.Correspondingly, it will be understood by those of skill in the art that the raising of NaCl in medium concentration is carried out on bacterial strain original salinity tolerance basis when the gradient domestication bacterial strain of Deng Yan territory, ocean environmental sources being carried out to high salinity tolerance.
The concrete grammar of described inflated with nitrogen Anaerobic culturel is: liquid nutrient medium is placed in culture vessel, and pass into nitrogen and the air in culture vessel is discharged completely, sealed vessel is cultivated afterwards.
Adopt the screening method of dissimilatory iron reduction bacterium of the present invention simple and easy to do and the screening cycle is short, without the need to the separating device of the costlinesses such as anaerobic operation system in screening process, effectively reduce screening cost, the purifying bacterial strain of acquisition has stronger Fe (III) reducing power.In addition, by dissimilatory iron reduction bacterial strain Cr (VI) heavy metal and different salt concn environment to tolerance can be obtained fast, easily, for the biological restoration under the environment such as heavy metal, salt territory provides germ plasm resource to the heavy metal of purifying bacterial strain and the gradient domestication of high salinity tolerance.
Embodiment
Be described in detail below in conjunction with the gradient acclimation method of specific embodiment to heavy metal further after the screening method of dissimilatory iron reduction bacterium of the present invention and screening and high salinity tolerance.
Embodiment 1
(1) mode progressively improving Fe (III) concentration in liquid nutrient medium is utilized to carry out enrichment culture to settling mud: the settling mud for inoculating is selected from freshet bed mud.During inoculation, appropriate mud sample being added Fe (III) starting point concentration is in the ironic citrate liquid nutrient medium of 10mmol/L, inflated with nitrogen Anaerobic culturel in serum bottle, before Anaerobic culturel, in substratum, in inflated with nitrogen to serum bottle, air carries out sealing cultivation again after discharging completely, after cultivating 48h, measure OD in liquid nutrient medium 600value and Fe (II) concentration, select OD 600value>=0.500 and the liquid nutrient medium of Fe (II) concentration>=1mmol/L, by supernatant bacterium liquid wherein by 1% inoculum size to be forwarded to Fe (III) concentration be in the ironic citrate liquid nutrient medium of 20mmol/L, select OD after cultivating 48h 600value>=0.500 and supernatant bacterium liquid in the liquid nutrient medium of Fe (II) concentration>=1mmol/L, again being forwarded to Fe (III) concentration is in the ironic citrate liquid nutrient medium of 30mmol/L, repeat aforesaid operations, each operation makes Fe (III) concentration in substratum improve 10mmol/L, until bring up to 50mmol/L.In enrichment process, culture condition is: pH7.0, temperature 26 DEG C.The concentration recording Fe in substratum (II) after enrichment culture terminates is 12mmol/L, shows that in mud sample, flora has obvious Fe3+ reduction ability.
(2) separation and purification dissimilatory iron reduction bacterium from enrichment flora: by the mud suspension after step (1) enrichment culture in the centrifugal 5min of 1000rpm, get supernatant bacterium liquid and dilute 100 times, get afterwards 100 μ L dilute after the streak inoculation of bacterium liquid in Fe (III) concentration be on the solid medium of 50mmol/L, the identical solid medium of one deck is toppled over again to ensure bacterium liquid and air exclusion after bacterium liquid is absorbed by solid medium completely, after the solid medium on upper strata solidifies, topple over the capping in addition of one deck whiteruss again, to guarantee the anaerobic culture environment of bacterium colony; This three layer flat plate is cultivated 48h, and culture condition is pH7.0, temperature 26 DEG C; Picking list bacterium colony afterwards, being inoculated in Fe (III) concentration is in the liquid nutrient medium of 50mmol/L, Anaerobic culturel 24h in serum bottle, and culture condition is pH7.0, temperature 26 DEG C; Cultivation terminates the concentration of Fe (II) in rear mensuration liquid nutrient medium, choose Fe (II) liquid nutrient medium that concentration is the highest, bacterium liquid is wherein inoculated in described three layer flat plate again and carries out cultivating and being separated, repeat this process 5 times, obtain purifying bacterial strain, correspondingly, record Fe in liquid nutrient medium (II) concentration and reach 18.5mmol/L.
The gradient domestication of heavy metal tolerance: bacterial strain step (2) obtained is inoculated in liquid nutrient medium containing Fe (III) (concentration is 50mmol/L), Anaerobic culturel in serum bottle by the inoculum size of 1%.Cultivate after 24h by bacterial strain by 1% inoculum size to be transferred to Cr (VI) concentration be in the liquid nutrient medium of 0.5mmol/L, cultivate one week OD in mensuration substratum afterwards 600the concentration of value and Fe (II), chooses OD 600the substratum of value>=0.500, Fe (II) concentration>=10mmol/L, by bacterial strain wherein by 1% inoculum size to be transferred to Cr (VI) concentration be in the liquid nutrient medium of 1.0mmol/L, repeat aforesaid operations, each operation makes Cr in substratum (VI) concentration improve 0.5mmol/L, until Cr (VI) concentration is increased to 2.0mmol/L.The concentration of the Cr (VI) that the bacterial strain obtained by above-mentioned gradient domestication can be tolerated reaches 1.5mmol/L, correspondingly, records Fe in liquid nutrient medium (II) concentration and reaches 6.7mmol/L.Liquid nutrient medium for adding Cr (VI) in this step is also the liquid nutrient medium containing Fe (III) (concentration is 50mmol/L), and culture condition is: pH7.0, temperature 26 DEG C.
The gradient domestication of high salinity tolerance: purifying bacterial strain step (2) obtained is inoculated in the liquid nutrient medium containing Fe (III) (concentration is 50mmol/L) by the inoculum size of 1%, Anaerobic culturel in serum bottle.During initial incubation, in substratum, do not add NaCl, after cultivating one week, by bacterial strain by 1% inoculum size to be transferred to NaCl concentration be in the liquid nutrient medium of 5g/L, cultivate after one week and measure substratum OD 600the concentration of value and Fe (II), chooses OD 600be worth>=0.500 and the substratum of Fe (II) concentration>=10mmol/L, by bacterial strain wherein by 1% inoculum size to be transferred to NaCl concentration be in the liquid nutrient medium of 10g/L, repeat aforesaid operations, each operation makes NaCl concentration in liquid nutrient medium improve 5g/L, until NaCl concentration is increased to 20g/L.The bacterial strain tolerance NaCl concentration obtained by above-mentioned gradient domestication reaches 20g/L, correspondingly, records Fe in liquid nutrient medium (II) concentration and reaches 9.8mmol/L.Liquid nutrient medium for adding NaCl in this step is also the liquid nutrient medium containing Fe (III) (concentration is 50mmol/L), and culture condition is: pH7.0, temperature 26 DEG C.
Embodiment 2
(1) mode progressively improving Fe (III) concentration in liquid nutrient medium is utilized to carry out enrichment culture to settling mud: the settling mud for inoculating is selected from the tideland bed mud of ocean offshore.Except enrichment culture condition, this step is identical to the enrichment culture mode of freshet bed mud with embodiment 1 step (1), and in this step, enrichment culture condition is: pH8.0, temperature 35 DEG C, and NaCl concentration is 25g/L.The concentration recording Fe in substratum (II) after enrichment culture terminates is 5.5mmol/L, shows that in mud sample, flora has obvious Fe3+ reduction ability.
(2) separation and purification dissimilatory iron reduction bacterium from enrichment flora: this step is substantially identical with the mode of separation and purification dissimilatory iron reduction bacterium in embodiment 1 step (2), difference is that the concentration of Fe (III) in the solid medium that adopts in this step and liquid nutrient medium is 10mmol/L, the training time in three layer flat plate and liquid nutrient medium is 48h, corresponding culture condition is pH8.0, temperature 35 DEG C, NaCl concentration is 25g/L.Repeatedly cultivate through 6 times and be separated, obtain purifying bacterial strain, correspondingly, record Fe in liquid nutrient medium (II) concentration and reach 8.2mmol/L.
The gradient domestication of heavy metal tolerance: the bacterial strain that step (2) is obtained by 1% inoculum size to be inoculated in Fe (III) concentration be the liquid nutrient medium of 10mmol/L, in serum bottle after Anaerobic culturel 24h by bacterial strain by 1% inoculum size to be transferred to Cr (VI) concentration be in the liquid nutrient medium of 0.5mmol/L, cultivate after one week and measure OD in substratum 600the concentration of value and Fe (II), chooses OD 600the liquid nutrient medium of value>=0.500, Fe (II) concentration>=2mmol/L, by bacterial strain wherein by 1% inoculum size to be transferred to Cr (VI) concentration be in the liquid nutrient medium of 1.0mmol/L, repeat aforesaid operations, each operation makes Cr in substratum (VI) concentration improve 0.5mmol/L, until Cr (VI) concentration is increased to 2.5mmol/L.The concentration of the Cr (VI) that the bacterial strain obtained by above-mentioned gradient domestication can be tolerated reaches 2.0mmol/L, correspondingly, records Fe in liquid nutrient medium (II) concentration and reaches 3.6mmol/L.Liquid nutrient medium for adding Cr (VI) in this step is also the liquid nutrient medium containing Fe (III) (concentration is 10mmol/L), and culture condition is: pH8.0, temperature 35 DEG C, and NaCl concentration is 25g/L.
The gradient domestication of high salinity tolerance: purifying bacterial strain step (2) obtained is inoculated in the liquid nutrient medium containing Fe (III) (concentration is 50mmol/L) by the inoculum size of 1%, Anaerobic culturel in serum bottle.The starting point concentration of NaCl in medium is 25g/L, after cultivating one week, by bacterial strain by 1% inoculum size to be transferred to NaCl concentration be in the liquid nutrient medium of 30g/L, cultivate after one week and measure substratum OD 600the concentration of value and Fe (II), chooses OD 600be worth>=0.500 and the substratum of Fe (II) concentration>=2mmol/L, by bacterial strain wherein by 1% inoculum size to be transferred to NaCl concentration be in the liquid nutrient medium of 35g/L, repeat aforesaid operations, each operation makes NaCl concentration in liquid nutrient medium improve 5g/L, until NaCl concentration is increased to 60g/L.The bacterial strain tolerance NaCl concentration obtained by above-mentioned gradient domestication reaches 60g/L, correspondingly, records Fe in liquid nutrient medium (II) concentration and reaches 2.6mmol/L.Liquid nutrient medium for adding NaCl in this step is also the liquid nutrient medium containing Fe (III) (concentration is 10mmol/L), and culture condition is: pH8.0, temperature 35 DEG C.
Embodiment 3
(1) mode progressively improving Fe (III) concentration in liquid nutrient medium is utilized to carry out enrichment culture to settling mud: the settling mud for inoculating is selected from oceanic sediment bed mud.Except enrichment culture condition, this step is identical to the enrichment culture mode of freshet bed mud with embodiment 1 step (1), and in this step, enrichment culture condition is: pH8.0, temperature 21 DEG C, and NaCl concentration is 30g/L.The concentration recording Fe in substratum (II) after enrichment culture terminates is 6mmol/L., show that in mud sample, flora has obvious Fe3+ reduction ability.
(2) separation and purification dissimilatory iron reduction bacterium from enrichment flora: this step is substantially identical with the mode of separation and purification dissimilatory iron reduction bacterium in embodiment 1 step (2), difference is that the concentration of Fe (III) in the solid medium that adopts in this step and liquid nutrient medium is 30mmol/L, the training time in three layer flat plate and liquid nutrient medium is 48h, corresponding culture condition is: pH8.0, temperature 21 DEG C, NaCl concentration is 30g/L.Repeatedly cultivate through 5 times and be separated, obtain purifying bacterial strain, correspondingly, record Fe in liquid nutrient medium (II) concentration and reach 24.3mmol/L.
The gradient domestication of heavy metal tolerance: the bacterial strain that step (2) is obtained by 1% inoculum size to be inoculated in Fe (III) concentration be in the liquid nutrient medium of 30mmol/L, in serum bottle after Anaerobic culturel 24h by bacterial strain by 1% inoculum size to be transferred to Cr (VI) concentration be in the liquid nutrient medium of 0.5mmol/L, cultivate after one week and measure OD in substratum 600the concentration of value and Fe (II), chooses OD 600the liquid nutrient medium of value>=0.500, Fe (II) concentration>=5mmol/L, by bacterial strain wherein by 1% inoculum size to be transferred to Cr (VI) concentration be in the liquid nutrient medium of 1.0mmol/L, repeat aforesaid operations, each operation makes Cr in substratum (VI) concentration improve 0.5mmol/L, until Cr (VI) concentration is increased to 2.5mmol/L.The concentration of the Cr (VI) that the bacterial strain obtained by above-mentioned gradient domestication can be tolerated reaches 2.0mmol/L, correspondingly, records Fe in liquid nutrient medium (II) concentration and reaches 23.5mmol/L.Liquid nutrient medium for adding Cr (VI) in this step is also the liquid nutrient medium containing Fe (III) (concentration is 10mmol/L), and culture condition is: pH8.0, temperature 21 DEG C, and NaCl concentration is 30g/L.
The gradient domestication of high salinity tolerance: the purifying bacterial strain that step (2) is obtained by 1% inoculum size to be inoculated in Fe (III) concentration be in the liquid nutrient medium of 30mmol/L, Anaerobic culturel in serum bottle.During initial incubation, the starting point concentration of NaCl in medium is 30g/L, after cultivating one week, by bacterial strain by 1% inoculum size to be transferred to NaCl concentration be in the liquid nutrient medium of 35g/L, cultivate after one week and measure substratum OD 600the concentration of value and Fe (II), chooses OD 600be worth>=0.500 and the substratum of Fe (II) concentration>=5mmol/L, by bacterial strain wherein by 1% inoculum size to be transferred to NaCl concentration be in the liquid nutrient medium of 40g/L, repeat aforesaid operations, each operation makes NaCl concentration in liquid nutrient medium improve 5g/L, until NaCl concentration is increased to 60g/L.The bacterial strain tolerance NaCl concentration obtained by above-mentioned gradient domestication reaches 60g/L, correspondingly, records Fe in liquid nutrient medium (II) concentration and reaches 11.5mmol/L.Liquid nutrient medium for adding NaCl in this step is also the liquid nutrient medium containing Fe (III) (concentration is 10mmol/L), and culture condition is: pH8.0, temperature 21 DEG C.

Claims (4)

1. screen a method for dissimilatory iron reduction bacterium, it is characterized in that comprising the steps:
(1) mode progressively improving Fe (III) concentration in liquid nutrient medium is utilized to carry out enrichment culture to settling mud: to be added by mud sample in the liquid nutrient medium containing Fe (III), inflated with nitrogen Anaerobic culturel, in described liquid nutrient medium, the starting point concentration of Fe (III) is 5 ~ 10mmol/L, flora stronger for Fe (III) reducing power is progressively forwarded in the higher liquid nutrient medium of Fe (III) concentration in culturing process, the concentration of Fe (III) is improved 5 ~ 10mmol/L, until bring up to 50mmol/L by each switching;
(2) separation and purification dissimilatory iron reduction bacterium from enrichment flora: by centrifugal for the mud suspension after step (1) enrichment culture, get supernatant bacterium liquid dilution after streak inoculation in Fe (III) concentration be on the solid medium of 10 ~ 50mmol/L, the identical solid medium of one deck is toppled over again after bacterium liquid is absorbed by solid medium completely, after the solid medium on upper strata solidifies, then topple over the capping in addition of one deck whiteruss; By picking list bacterium colony after this three layer flat plate cultivation 24 ~ 72h, being inoculated in Fe (III) concentration is in the liquid nutrient medium of 10 ~ 50mmol/L, Fe in liquid nutrient medium (II) concentration is measured after inflated with nitrogen Anaerobic culturel 24 ~ 72h, choose accumulation Fe (II) bacterial strain that concentration is the highest, again be inoculated in described three layer flat plate and carry out cultivating and being separated, repeat said process, until obtain purifying bacterial strain;
In described step (1), (2), the potential of hydrogen of substratum all remains on: pH6.0 ~ 9.0; Corresponding culture temperature is: 20 ~ 40 DEG C.
2. the method for screening dissimilatory iron reduction bacterium according to claim 1, is characterized in that: the gradient domestication step also comprising heavy metal tolerance or high salinity tolerance after described step (2); The gradient domestication step of described heavy metal tolerance takes following method: purifying inoculation step (2) obtained is in the liquid nutrient medium of 10 ~ 50mmol/L in Fe (III) concentration, inflated with nitrogen Anaerobic culturel; In culturing process, heavy metal tolerance and the stronger bacterial strain of Fe (III) reducing power are progressively forwarded in the higher liquid nutrient medium of Cr (VI) concentration, in liquid nutrient medium, the maximum concentration of Cr (VI) is no more than 2.5mmol/L, the concentration of Fe (III) remains unchanged, and substratum potential of hydrogen all remains on: pH6.0 ~ 9.0; Corresponding culture temperature is: 20 ~ 40 DEG C;
Described high salinity tolerance gradient domestication step take following method: the purifying inoculation that step (2) is obtained in Fe (III) concentration be inflated with nitrogen Anaerobic culturel in the liquid nutrient medium of 10 ~ 50mmol/L, in culturing process, salt resistance ability and the stronger bacterial strain of Fe (III) reducing power are progressively forwarded in the higher liquid nutrient medium of NaCl concentration, in liquid nutrient medium, the maximum concentration of NaCl is no more than 60g/L, the concentration of Fe (III) remains unchanged, and substratum potential of hydrogen all remains on: pH6.0 ~ 9.0; Corresponding culture temperature is: 20 ~ 40 DEG C.
3. the method for screening dissimilatory iron reduction bacterium according to claim 1 and 2, is characterized in that: the liquid nutrient medium containing Fe (III) used in the gradient domestication step of described step (1), (2), heavy metal tolerance and high salinity tolerance is ironic citrate substratum; The solid medium containing Fe (III) used in described step (2) is ironic citrate solid medium.
4. the method for screening dissimilatory iron reduction bacterium according to claim 1 and 2, it is characterized in that: in the gradient domestication step of described step (1), (2), heavy metal tolerance and high salinity tolerance, the method of inflated with nitrogen Anaerobic culturel is: liquid nutrient medium is placed in culture vessel, passing into nitrogen makes the air in culture vessel discharge completely, and sealed vessel is cultivated afterwards.
CN201510183645.8A 2015-04-17 2015-04-17 Method for screening dissimilatory Fe(III)-reducing bacteria Pending CN104745519A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106544273A (en) * 2016-10-25 2017-03-29 泰州学院 A kind of alienation Fe (III) reduces multi strain co cultivation method
CN108077048A (en) * 2017-11-03 2018-05-29 东莞理工学院 A kind of method of iron-reducing bacterium reduction plant root border cells
CN108911120A (en) * 2018-07-18 2018-11-30 山西龙盘微生物科技有限公司 A kind of acclimation method for daily use chemicals cleaning product activated sludge of degrading
CN109019822A (en) * 2018-08-07 2018-12-18 北京伦至环境科技有限公司 A kind of Organic Chemicals In Groundwater in-situ remediation method
CN109248913A (en) * 2017-07-14 2019-01-22 武汉科技大学 A kind of method that original inhabitants iron bacteria solidifies rufous basalt residual soil
CN109851059A (en) * 2019-01-24 2019-06-07 天津科技大学 The method for preparing biological magnetic iron ore removal heavy metal chromium using dissimilatory iron reduction bacterium
CN110699296A (en) * 2019-11-12 2020-01-17 黑龙江八一农垦大学 Iron reduction complex microbial inoculant and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104458624A (en) * 2014-12-19 2015-03-25 中国环境科学研究院 Method for analyzing electron transfer capability of in-situ humus of soil

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104458624A (en) * 2014-12-19 2015-03-25 中国环境科学研究院 Method for analyzing electron transfer capability of in-situ humus of soil

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
李顺鹏主编: "《微生物学实验指导》", 31 January 2015 *
王亚娥等: "不同Fe(III)对活性污泥异化铁还原及除磷影响研究", 《中国环境科学》 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106544273A (en) * 2016-10-25 2017-03-29 泰州学院 A kind of alienation Fe (III) reduces multi strain co cultivation method
CN109248913A (en) * 2017-07-14 2019-01-22 武汉科技大学 A kind of method that original inhabitants iron bacteria solidifies rufous basalt residual soil
CN108077048A (en) * 2017-11-03 2018-05-29 东莞理工学院 A kind of method of iron-reducing bacterium reduction plant root border cells
CN108911120A (en) * 2018-07-18 2018-11-30 山西龙盘微生物科技有限公司 A kind of acclimation method for daily use chemicals cleaning product activated sludge of degrading
CN109019822A (en) * 2018-08-07 2018-12-18 北京伦至环境科技有限公司 A kind of Organic Chemicals In Groundwater in-situ remediation method
CN109851059A (en) * 2019-01-24 2019-06-07 天津科技大学 The method for preparing biological magnetic iron ore removal heavy metal chromium using dissimilatory iron reduction bacterium
CN109851059B (en) * 2019-01-24 2022-04-08 天津科技大学 Method for removing heavy metal chromium from biological magnetite prepared by using dissimilatory iron reducing bacteria
CN110699296A (en) * 2019-11-12 2020-01-17 黑龙江八一农垦大学 Iron reduction complex microbial inoculant and application thereof

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