CN109266569A - A kind of method and its application separating efficient anaerobic arsenic reducing bacteria - Google Patents
A kind of method and its application separating efficient anaerobic arsenic reducing bacteria Download PDFInfo
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- CN109266569A CN109266569A CN201810948341.XA CN201810948341A CN109266569A CN 109266569 A CN109266569 A CN 109266569A CN 201810948341 A CN201810948341 A CN 201810948341A CN 109266569 A CN109266569 A CN 109266569A
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- arsenic
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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
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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
- C12N1/02—Separating microorganisms from their culture media
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Abstract
The invention discloses a kind of methods for separating efficient anaerobic arsenic reducing bacteria, are included in basin drilling 13-15m, take deposit, and deposit is added in LB culture medium, cultivate under the conditions of 32 DEG C；Suspension is taken in LB culture medium, and is inoculated in the fresh culture containing As (V), 3 times repeatedly；It takes bacterium solution to dilute, the bacterium solution after dilution is uniformly applied on the LB solid medium containing corresponding As (V) concentration with the method that plate is coated with；It is placed in 32 DEG C of constant incubators and cultivates, shaped to bacterium colony；Picking single colonie is purified using the method that plate streaking separates to get efficient anaerobic arsenic reducing bacteria is arrived.The bacterium that the present invention obtains has very strong reducing power to As (V), can effectively facilitate arsenic from sediment transport to water body in, improve arsenic the amount of migration twice or more.
Description
Technical field
The present invention relates to field of environment microorganism more particularly to a kind of method for separating efficient anaerobic arsenic reducing bacteria and its answer
With.
Background technique
Arsenic is the toxic metalloid being widely present in nature.Due to being taken in for a long time containing As groundwater, the whole world is tens million of
People faces internal organs cancer and dermopathic threat.Although arsenic is toxic to many biologies, some bacteriums can live in height
In As groundwater environment, and play an important role in the geochemical behaviour of arsenic.Microorganism is in long-term Historical Evolution process
In evolved various anti-arsenic mechanism, wherein it is most important have cytoplasm arsenic reduction and alienation arsenic reduction.Studies have shown that in environment
The arsenic reduction of microorganism is the major reason for causing water body arsenic pollution to be aggravated.For example some arsenic also pathogenic microorganism can will adsorb
As (V) on mineral or deposit is reduced to the As (III) with mobility and strong toxicity, this discharges arsenic from solid phase
It has arrived in liquid phase, so as to cause the arsenic pollution at the water sources such as underground water.Research about arsenic also pathogenic microorganism is to environment As pollution
Problem is of great significance, but current research focuses primarily upon the geochemical cycle of the arsenic of aerobic microbiological participation, about
The research of arsenic anaerobe is started late, and Research foundation is relatively weak.
Summary of the invention
In view of this, the embodiment provides a kind of method and its application for separating efficient anaerobic arsenic reducing bacteria.
The embodiment of the present invention provides a kind of method for separating efficient anaerobic arsenic reducing bacteria, comprising the following steps:
S1. it drills in basin to 13-15m, takes deposit, wrapped up and be sealed in freshness protection package with preservative film, and quickly transported
It returns；It prepares 100ml LB culture medium (g/L): yeast extract 5g, peptone 10g, sodium chloride 5g, in 121 DEG C of high-temperature sterilizations
10min, it is cooling stand-by；It selects the deposit after rejecting outer layer in anaerobic culture box to be added in sterile LB medium, deposit
Mass volume ratio with LB culture medium is 1:10, is protected from light culture under the conditions of 32 DEG C after shaking up, obtains mixed-culture medium；
S2. after cultivating 72h, 0.1mL suspension is drawn from the mixed-culture medium of standing, is inoculated in 100mL containing 1200mg/
In the fresh culture of L As (V), 3 times repeatedly；
S3. 1mL bacterium solution is taken to be diluted to 10 from fresh culture-6, the method that the bacterium solution after dilution is coated with plate is equal
It is even to be applied on the LB solid medium containing corresponding As (V) concentration, the LB solid medium 5g/L containing yeast extract,
Peptone 10g/L, sodium chloride 5g/L, agar 20g/L；
S4. it is placed in 32 DEG C of constant incubators and cultivates, shaped to bacterium colony；
S5. picking single colonie is purified using the method that plate streaking separates to get efficient anaerobic arsenic reducing bacteria is arrived.
Further, in the step S5, single colonie oese picking.
Further, in the step S5, single strain after purification, which is placed in paraffin, to be saved.
A kind of application of efficient anaerobic arsenic reducing bacteria, the efficient anaerobic arsenic reducing bacteria under anaerobic, can restore for 4 days
80% or more As (V).
A kind of application of efficient anaerobic arsenic reducing bacteria, the efficient anaerobic arsenic reducing bacteria can promote arsenic from sediment transport
Into water body, make twice of the raising of arsenic the amount of migration or more.
Compared with prior art, the present invention has the effect that the bacterium that separation obtains has very strong go back to As (V)
Proper energy power, can effectively facilitate arsenic from sediment transport to water body in, improve arsenic the amount of migration twice or more.
Detailed description of the invention
Fig. 1 is a kind of flow chart for the method for separating efficient anaerobic arsenic reducing bacteria of the present invention.
Fig. 2 is the Phylogenetic figure of efficient anaerobic arsenic reducing bacteria Bacillus sp.XZM of the invention.
The growth curve in As (V) that Fig. 3 is efficient anaerobic arsenic reducing bacteria Bacillus sp.XZM of the invention is special
Sign figure and As (V) also original image.
Fig. 4 is that efficient anaerobic arsenic reducing bacteria Bacillus sp.XZM of the invention migrates arsenic situation explanation from deposit
Figure.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to embodiment party of the present invention
Formula is further described.
Embodiment 1
Referring to FIG. 1, the embodiment provides a kind of method for separating efficient anaerobic arsenic reducing bacteria, including it is following
Step:
S1. it drills in basin to 13-15m, takes deposit, wrapped up and be sealed in freshness protection package with preservative film, and quickly transported
Go back to laboratory.It prepares 100ml LB culture medium (g/L): yeast extract 5g, peptone 10g, sodium chloride 5g, in 121 DEG C of high temperature
Sterilize 10min, cooling stand-by.The deposit after rejecting outer layer is selected in anaerobic culture box to be added in sterile LB medium,
In, the mass volume ratio of deposit and culture medium is 1:10, is protected from light culture under the conditions of 32 DEG C after shaking up, is mixed
Liquid；
S2. after cultivating 72h, 0.1mL suspension is drawn from the mixed-culture medium of standing, is inoculated in 100mL containing 1200mg/
In the fresh culture of L As (V), 3 times repeatedly；
S3. 1mL bacterium solution is taken to be diluted to 10 from fresh culture-6Afterwards, the method bacterium solution after dilution being coated with plate
It is uniformly applied on the LB solid medium containing corresponding As (V) concentration, culture medium 5g/L containing yeast extract, peptone
10g/L, sodium chloride 5g/L, agar 20g/L；
S4. it is placed in 32 DEG C of constant incubators and cultivates, shaped to bacterium colony；
S5. oese picking single colonie is used, is purified using the method that plate streaking separates to get efficient anaerobic arsenic is arrived
Reducing bacteria.
Single strain after purification, which is placed in paraffin, to be saved.
A kind of application of efficient anaerobic arsenic reducing bacteria, the efficient anaerobic arsenic reducing bacteria under anaerobic, can restore for 4 days
80% or more As (V).
A kind of application of efficient anaerobic arsenic reducing bacteria, the efficient anaerobic arsenic reducing bacteria can promote arsenic from sediment transport
Into water body.
The present invention has isolated one plant of anaerobism arsenic reducing bacteria using simple method, can quickly restore the As in water body
(V), can effectively facilitate arsenic from sediment transport to water body in, improve arsenic the amount of migration, experimental group arsenic migration in 6 days twice or more
Amount is 80.88 μ g/L (control group is 33.74 μ g/L).
The identification of efficient anaerobic arsenic reducing bacteria:
It extracts bacterial genomes DNA according to Ezup pillar bacterial genomes DNA extraction agent box (raw work) specification and is used for
PCR amplification, using strain idenfication universal primer, as shown in table 1:
The primer of 1 16S rDNA gene of table
To extract genomic DNA as template, PCR amplification 16S rDNA sequence, amplification system (table 2) and amplification program (table
3) as follows:
Table 2
Table 3
The band of 1.5Kb or so has been obtained, this band has been subjected to recovery purifying, and deliver sequencing.Sequencing result carries out
The BLAST of line is compared, and using the phylogenetic tree of Mage5 software building bacterial strain, and as a result as shown in Fig. 2, which is bud
Spore Bacillus.
The anaerobic reduction feature of efficient anaerobic arsenic reducing bacteria:
Bacillus sp.XZM is cultivated in LB culture medium to logarithmic phase, is inoculated into and contains respectively according to 1% inoculum concentration
It is primary per sampling for 24 hours in the LB culture medium of 0.25mM As (V), take 3ml to survey OD with ultraviolet specrophotometer600Value, takes 3ml mistake
0.22 μm of filter membrane, filter liquor with after SPE anion exchange post separation As (V) and As (III), with atomic fluorescence spectrophotometer (Ji Tian,
China) survey arsenic content.
The growth curve feature and As (V) reduction in As (V) of efficient anaerobic arsenic reducing bacteria Bacillus sp.XZM
As shown in Figure 3.It can be seen from the figure that bacterial strain to the reduction of As (V) since the logarithmic phase of cell, be computed, which can be
The As (V) of 80% or more reduction in 4 days.
Efficient anaerobic arsenic reducing bacteria promotes arsenic in deposit to discharge:
Bacillus sp.XZM is cultivated in LB culture medium to logarithmic phase, in anaerobic operation case according to 1% inoculation
Amount is inoculated into soil: water is in the microcosm device of 1:10, and mixing is placed on 180r/min oscillation training in 32 DEG C of thermostat water bath
It supports, while the control group of not inoculated bacteria is set.It is primary per sampling for 24 hours, total arsenic is surveyed with atomic fluorescence spectrophotometer (Ji Tian, China)
Content.
It is as shown in Figure 4 that efficient anaerobic arsenic reducing bacteria Bacillus sp.XZM migrates arsenic situation explanation from deposit.
In the absence of conflict, the feature in embodiment and embodiment herein-above set forth can be combined with each other.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (5)
1. a kind of method for separating efficient anaerobic arsenic reducing bacteria, which comprises the following steps:
S1. it drills in basin to 13-15m, takes deposit, wrapped up and be sealed in freshness protection package with preservative film, and quickly transported back；Match
100ml LB culture medium (g/L) processed: yeast extract 5g, peptone 10g, sodium chloride 5g are cold in 121 DEG C of high-temperature sterilization 10min
Stand-by；It selects the deposit after rejecting outer layer in anaerobic culture box to be added in sterile LB medium, deposit and LB are cultivated
The mass volume ratio of base is 1:10, is protected from light culture under the conditions of 32 DEG C after shaking up, obtains mixed-culture medium；
S2. after cultivating 72h, 0.1mL suspension is drawn from the mixed-culture medium of standing, is inoculated in 100mL As containing 1200mg/L
(V) in fresh culture, 3 times repeatedly；
S3. 1mL bacterium solution is taken to be diluted to 10 from fresh culture-6, the bacterium solution after dilution is uniformly applied with the method that plate is coated with
Smear in containing corresponding As (V) concentration LB solid medium on, the LB solid medium 5g/L containing yeast extract, albumen
Peptone 10g/L, sodium chloride 5g/L, agar 20g/L；
S4. it is placed in 32 DEG C of constant incubators and cultivates, shaped to bacterium colony；
S5. picking single colonie is purified using the method that plate streaking separates to get efficient anaerobic arsenic reducing bacteria is arrived.
2. the method for separation efficient anaerobic arsenic reducing bacteria according to claim 1, which is characterized in that in the step S5,
Single colonie oese picking.
3. the method for separation efficient anaerobic arsenic reducing bacteria according to claim 1, which is characterized in that in the step S5,
Single strain after purification, which is placed in paraffin, to be saved.
4. a kind of application of efficient anaerobic arsenic reducing bacteria, which is characterized in that the efficient anaerobic arsenic reducing bacteria under anaerobic,
80% or more As (V) can be restored within 4 days.
5. a kind of application of efficient anaerobic arsenic reducing bacteria, which is characterized in that the efficient anaerobic arsenic reducing bacteria can promote arsenic from
Sediment transport makes twice of the raising of arsenic the amount of migration or more into water body.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010048852A1 (en) * | 1998-06-18 | 2001-12-06 | Chowdhury Ajit K. | Stabilization of arsenic-contaminated materials |
| WO2002048335A2 (en) * | 2000-12-13 | 2002-06-20 | University Of Georgia Research Foundation, Inc. | Metal resistant plants and use for phytoremediation |
| CN101993835A (en) * | 2010-05-27 | 2011-03-30 | 威远川奇生工科技有限责任公司 | Arsenic-resistant bacteria and method for performing oxidation treatment on high-arsenic gold concentrate by using same |
| CN103013868A (en) * | 2012-12-04 | 2013-04-03 | 南京大学 | Sulfate reducing bacteria with tolerance on arsenic |
| CN105936884A (en) * | 2016-07-07 | 2016-09-14 | 中国地质大学（武汉） | Bosea sp. AS-1 strain capable of tolerating pollution of arsenic and antimony and oxidizing As(III) and applications thereof |
| CN108293345A (en) * | 2017-01-12 | 2018-07-20 | 喜施倍全球股份有限公司 | Microbe soil enhances |
-
2018
- 2018-08-20 CN CN201810948341.XA patent/CN109266569A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010048852A1 (en) * | 1998-06-18 | 2001-12-06 | Chowdhury Ajit K. | Stabilization of arsenic-contaminated materials |
| WO2002048335A2 (en) * | 2000-12-13 | 2002-06-20 | University Of Georgia Research Foundation, Inc. | Metal resistant plants and use for phytoremediation |
| CN101993835A (en) * | 2010-05-27 | 2011-03-30 | 威远川奇生工科技有限责任公司 | Arsenic-resistant bacteria and method for performing oxidation treatment on high-arsenic gold concentrate by using same |
| CN103013868A (en) * | 2012-12-04 | 2013-04-03 | 南京大学 | Sulfate reducing bacteria with tolerance on arsenic |
| CN105936884A (en) * | 2016-07-07 | 2016-09-14 | 中国地质大学（武汉） | Bosea sp. AS-1 strain capable of tolerating pollution of arsenic and antimony and oxidizing As(III) and applications thereof |
| CN108293345A (en) * | 2017-01-12 | 2018-07-20 | 喜施倍全球股份有限公司 | Microbe soil enhances |
Non-Patent Citations (3)
| Title |
|---|
| MARCELLA RUTA等: "As(V)-reduction to As(III) by arsenicresistant Bacillus spp. bacterial strains isolated from low-contaminated sediments of the Oliveri-Tindari Lagoon, Italy", 《CHEMISTRY AND ECOLOGY》 * |
| XIAOMING CHEN等: "Microbial communities involved in arsenic mobilization and release from the deep sediments into groundwater in Jianghan plain, Central China", 《SCIENCE OF THE TOTAL ENVIRONMENT》 * |
| 李冰等: "高砷地下水砷还原菌的分离鉴定", 《中国科技论文在线》 * |
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Application publication date: 20190125 |