CN109504625B - Bacillus cereus DIF1 and microbial agent produced by same and application of bacillus cereus DIF1 - Google Patents
Bacillus cereus DIF1 and microbial agent produced by same and application of bacillus cereus DIF1 Download PDFInfo
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Abstract
The invention discloses bacillus cereus DIF1, a microbial agent produced by the same and application of the bacillus cereus DIF1, wherein the strain is preserved in China center for type culture Collection in 2018, 5 months and 15 days, and the preservation number is CCTCC NO: m2018274. The bacillus cereus DIF1 has a strong dissimilatory reduction function of Fe (III), can efficiently reduce soluble Fe (III) and solid Fe (III), and has strong hexavalent chromium reduction capacity and electricity generation capacity simultaneously. The invention discloses a new property of bacillus cereus and application research in the aspect of functions thereof, and provides a new material for dissimilatory iron reducing bacteria in the aspect of treatment of environmental pollutants.
Description
Technical Field
The invention relates to environmental bioremediation, in particular to bacillus cereus DIF1, a microbial agent produced by the same and application of the microbial agent.
Background
Iron is one of the elements which are distributed on the earth most widely, is the element with the fourth highest abundance in the earth crust, and is one of the key factors of geological processes and environmental changes such as heavy metal migration and transformation and the migration of other organic pollutants. Microorganisms are the core players of the earth's surface iron cycle, and catabolism, which is characterized by the extracellular occurrence of iron oxidation or reduction reactions by bacteria, is an important driving force for the iron cycle. Microbial dissimilatory Fe (iii) reduction with extracellular iron oxide as the terminal electron acceptor is probably the earliest form of microbial metabolism.
The dissimilatory iron reducing bacteria are a general name of a microorganism which can take Fe (III) as a unique electron acceptor, reduce Fe (III), oxidize an organic carbon source and obtain energy from the organic carbon source for self growth. The dissimilatory iron reducing bacteria can reduce various metal ions in the process of breathing and growing of bacteria, remove organic matters and heavy metals, and transfer electrons metabolized by intracellular organic matters to an extracellular electron acceptor, such as solid metal oxide, through an extracellular electron transfer mechanism to reduce the metal oxide. Therefore, the dissimilatory iron-reducing bacteria play an important role in the geochemical cycle of metal elements, the recovery and utilization of energy, and the like.
Chromium pollution is one of the most common heavy metal pollutants at present, and certain concentration of Cr (VI) can threaten life and health. Chromium is mainly used in industries such as metal processing, electroplating, tanning and the like, and chromate is often added in the industrial production process to prevent circulating water from corroding equipment. The waste water and waste gas discharged by industrial department are artificial sources of chromium in environment. Although chromium in industrial wastewater is mainly present in trivalent form, trivalent chromium discharged into the natural environment is converted into more toxic hexavalent chromium under environmental influence. Chromium in the water environment generally exists in the forms of Cr (VI) and Cr (III), and Cr (VI) has strong fluidity and can cause cancer due to chronic poisoning after being exposed to hexavalent chromium for a long time. The method for reducing chromium is chemical reduction, adsorption, ion exchange, membrane separation, bioremediation and electrochemical remediation, wherein the bioremediation technology provides a means without secondary pollution for chromium pollution areas.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide bacillus cereus DIF1, which has a reduction effect on iron ions and chromium ions and can be applied to recycling of iron and repairing environmental pollution caused by heavy metal ions. The bacillus cereus DIF1 has good electricity generating capacity and can be used for microbial fuel cells.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides bacillus cereus DIF1, which is preserved in the China center for type culture Collection with the preservation time of 2018, 5 and 15 days and the preservation number of CCTCCNO: m2018274.
The bacillus cereus DIF1 is a gram-positive bacterium, the bacterium is rod-shaped, the end is square, the bacterial colony is gray circle-shaped, the optimal growth temperature is 30 ℃, and the optimal growth pH value is 7.
In a second aspect, the present invention provides the use of Bacillus cereus DIF1 for dissimilatory reduction of Fe (III).
In a third aspect, the present invention provides the use of Bacillus cereus DIF1 for the reduction of hexavalent chromium.
In a fourth aspect, the present invention provides a microbial agent produced by bacillus cereus DIF1, said microbial agent being produced by fermentation of bacillus cereus DIF 1.
The fermentation medium is LB liquid medium which comprises 8-12g/L NaCl, preferably 10g/L, 4-6g/L yeast extract, preferably 5g/L, 8-12g/L peptone, preferably 10g/L, and pH 6.8-7.2, preferably 7.2.
In a fifth aspect, the present invention provides a method for culturing bacillus cereus DIF1, which comprises inoculating bacillus cereus DIF1 in LB liquid medium for shake culture.
The temperature of the shaking culture is 28-32 ℃, preferably 30 ℃, and the rotation speed is 100-300rmp, preferably 120 rpm.
In a sixth aspect, the invention provides the use of a microbial agent prepared from bacillus cereus DIF1 or bacillus cereus DIF1 in a microbial fuel cell.
Has the advantages that: compared with the prior art, the bacillus cereus DIF1 is obtained by separation, and has stronger reducing function of dissimilatory reducing Fe (III); bacillus cereus DIF1 is also effective in reducing Cr (VI) ions; the bacillus cereus DIF1 can be applied to a microbial fuel cell as an electricity generating bacterium; the invention widens the application and research thought of Bacillus cereus in the aspect of functions, provides a new material for dissimilatory iron reducing bacteria in the aspect of treatment of environmental pollutants, and has strong practical application value.
Drawings
FIG. 1 is a colony electron micrograph of Bacillus cereus DIF 1;
FIG. 2 is a 16SrDNA electrophoretogram of Bacillus cereus DIF 1;
FIG. 3 is a colony of Bacillus cereus DIF 1;
FIG. 4 is a phylogenetic tree of Bacillus cereus DIF1 based on the 16SrDNA sequence;
FIG. 5 is a graph of the iron reduction rate of Bacillus cereus DIF1 in an experiment for reducing ferric citrate;
FIG. 6 is a graph showing the iron reduction rate of Bacillus cereus DIF1 in an experiment for reducing iron oxide;
FIG. 7 is a graph of Bacillus cereus DIF1 in reducing Cr (VI);
FIG. 8 is a time power curve of Bacillus cereus DIF1 in a microbial fuel cell.
Detailed Description
The technical solution of the present invention is further explained below with reference to the embodiments and the accompanying drawings.
Example 1: isolation, screening and characterization of strains
1.1 Experimental articles
The experimental sample was taken from the underground iron ore soil of meishan iron ore.
DM culture medium: NaHCO 23 2.5g/L、CaCl2 0.08g/L、NH4Cl 1.0g/L、MgCl2·6H2O 0.2g/L、NaCl 10g/L、HEPES 7.2g/L, yeast extract 0.5g/L, lactic acid 0.9g/L, Wolfe's vitamin solution (Wolfe's vitamin solution)1mL/L, Wolfe's mineral solution (Wolfe's mineral solution)10mL/L, pH7.2, and both the Wolfe's vitamin solution and the Wolfe's mineral solution were added to the autoclaved DM solution through a sterile filter.
LB liquid medium: NaCl 10g/L, yeast extract 5g/L, peptone 10g/L, pH 7.2.
LB solid medium: NaCl 10g/L, yeast extract 5g/L, peptone 10g/L, agar 15g/L, pH7.2.
Electrolyte in cathode chamber of cell: 50mmol/L potassium ferricyanide, 40mmol/L potassium dihydrogen phosphate and 60mmol/L dipotassium hydrogen phosphate.
1.2 Dual Chamber Microbial Fuel Cell (MFC) Assembly and Start-Up
The bipolar chamber microbial fuel cell is constructed according to the following steps:
(1) placing the prepared DM culture medium, the treated carbon sheet electrode, the proton exchange membrane, the customized bipolar chamber glass bottle tightly wrapped by newspaper and the rubber plug into an autoclave for sterilization at 121 ℃ for 30 min;
(2) placing the sterilized experimental material into a clean bench for ultraviolet sterilization for 20 min;
(3) the two electrode chambers are connected by the silicon rubber and the proton exchange membrane, and are clamped by iron clamps to prevent falling off;
(4) 100mL of 50mmol/L potassium ferricyanide solution is poured into the cathode chamber, 20g of fine iron ore soil is filled into the anode chamber, and 100mL of DM culture solution is poured into the anode chamber;
(5) an electrode is arranged in the anode chamber, the anode chamber is sealed by a rubber plug and a sealing film, a small hole is reserved in the cathode chamber so as to facilitate the introduction of oxygen, and the battery is wrapped by tinfoil;
(6) the assembled bipolar chamber was placed in a constant temperature incubator, and the temperature was set to 25 ℃. The battery is externally connected with a 5k omega resistor, and the output voltage of the battery is collected by adopting a 30-channel USB data acquisition card of Beijing Ruibo Hua company. The DM culture medium and the potassium ferricyanide solution are periodically replaced every week to ensure that the battery stably operates, and the time interval is one operation period.
1.3 isolation and screening of the strains
Taking out the electrodes from the successfully started batteries on a sterile operating platform, picking the bacteria from the bacteria dense position by using a bacteria picking rod, coating streaks on the prepared solid LB culture medium, putting the solid LB culture medium into a constant-temperature incubator at 30 ℃, and culturing overnight. And selecting colonies which singly grow on the surface of the LB solid medium, streaking on a new LB solid medium, and culturing overnight. This was repeated several times until the growth morphology of the bacteria was stable, as shown in FIG. 3. The purified strain was identified (see below) as bacillus cereus and deposited in the chinese type culture collection at 2018, 5, 15, at the address of the collection unit: the preservation number of Wuhan university in Wuhan, China is CCTCCNO: m2018274.
The stable culture dish is stored in a refrigerator at 4 ℃ for later use, meanwhile, a bacterial liquid obtained by shaking the bacteria with an LB liquid culture medium (30 ℃, 120rpm) is mixed with 60 percent of sterilized glycerol 1:1, and the mixture is put in the refrigerator at minus 80 ℃ for freezing storage.
1.4 Strain characterization and identification
(1) The colony morphology of the strain is as follows: the bacterial colony is gray white and round in LB solid culture medium, as shown in figure 3, gram stain shows that the bacteria are rod-shaped and have the end square, as shown in figure 1, gram-positive bacteria, the optimal growth temperature is 30 ℃, and the optimal growth pH value is 7.
(2) Identifying 16SrDNA gene molecules of the strain: the nuclear DNA of the bacteria is used as a template, the universal primer for PCR amplification of the 16S rDNA gene is used as a primer, PCR amplification is carried out, an amplification band with the length of 1418bp is obtained (detected by 1% agarose gel electrophoresis), and as shown in figure 2, the whole sequence of the PCR product is determined after the PCR product is purified. The results are shown in SEQ ID No: 1, the length of the 16SrDNA gene sequence is 1418bp, the 16SrDNA gene sequence is compared with a GenBank database by a BLAST analysis method, the genetic relationship between the strain and Bacillus cereus is the closest, the homology is more than 99%, the strain is named as Bacillus cereus DIF1, and the phylogenetic evolution tree of the Bacillus cereus DIF1 based on the 16SrDNA gene sequence is shown in figure 4.
Example 2: application of Bacillus cereus (Bacillus cereus) DIF1 in reduction of Fe (III)
2.1 experiments with Bacillus cereus DIF1 for reducing ferric citrate:
preparing DM solution, adding 2mmol/L ferric citrate into the solution, sterilizing at high temperature, adding into sterile 100ml serum bottle, adding bacteria solution according to the inoculation concentration of 2.1 part, and sealing with the inoculation concentration of 107and/L, in the control experiment, the Bacillus cereus DIF1 bacterial liquid is not added, and the mixture is placed in a constant temperature incubator at the temperature of 30 ℃ for dark culture. Extracting the sample once every 12h in a sterile operation table, and respectively detecting Fe in the sample by using an ultraviolet spectrophotometer according to a method for detecting an iron reduction standard curve2+The results of the concentration change and the bacterial growth curve (OD600) are shown in FIG. 5, and the results show that the iron reduction capacity of the Bacillus cereus DIF1 is about 0.55mmol/L in 120h, while the control experiment only has trace Fe2+And (4) generating.
2.2 reduction of iron oxide by Bacillus cereus DIF 1:
preparing DM solution, and adding 100mg/100ml of ferric oxide into the solution. Sterilizing at high temperature, adding into sterile 100ml serum bottle, adding bacterial liquid according to the inoculation concentration of 2.1 part, sealing, adding Bacillus cereus DIF1 bacterial liquid in control experiment, and culturing in 30 deg.C constant temperature incubator in dark light. Extracting the sample once every 12h in a sterile operating platform, and respectively detecting Fe in the sample by using an ultraviolet spectrophotometer2+The results of the concentration change and the bacterial growth curve (OD600) are shown in FIG. 6, and the results show that the iron reduction capacity of the Bacillus cereus DIF1 is about 0.62mmol/L in 96h, while the control experiment only has trace Fe2+The result shows that Bacillus cereus (Bacillus cereus) DIF1 has dissimilatory iron reducing ability.
From the above experimental results, it can be seen that the Bacillus cereus of the present invention efficiently reduces fe (iii) in ferric citrate and ferric oxide, indicating that the Bacillus cereus (Bacillus cereus) DIF1 of the present invention has a strong reducing function of dissimilating and reducing fe (iii).
Example 3: application of Bacillus cereus (Bacillus cereus) DIF1 in hexavalent chromium reduction
Bacillus cereus (Bacillus cereus) DIF1 test for Cr (VI) reduction capability:
preparing DM solution, adding 2mmol/L anhydrous sodium chromate into the solution, sterilizing at high temperature, adding into a sterile 100ml serum bottle, adding Bacillus cereus (Bacillus cereus) DIF1 bacterial liquid according to the inoculation amount of 2.1 parts, sealing, adding no bacterial liquid in the control experiment, and performing dark culture in a 30 ℃ constant temperature incubator. Every 12h, extracting a sample in a sterile operation table, and respectively detecting Cr of the sample by using an ultraviolet spectrophotometer according to a method for measuring a chromium reduction standard curve6+The results of the concentration change and the bacterial growth curve (OD600) are shown in FIG. 7, and Cr is between 0 and 24 hours6+The concentration is reduced rapidly, the chromium reduction capability of the Bacillus cereus (Bacillus cereus) DIF1 in 96h is about 1.44mmol/L, and the Cr reduction capability of a control experiment is Cr6+The concentration is almost unchanged, which indicates that the Bacillus cereus DIF1 has better chromium reduction capability.
Example 4: application of Bacillus cereus (Bacillus cereus) DIF1 in microbial fuel cell
Bacillus cereus (Bacillus cereus) DIF1 is inoculated according to the concentration of 2.1 part, and is placed on the anode of the double-chamber microbial fuel cell constructed according to the 1.2 part of the invention to determine a power density curve, and the output power density of the microbial fuel cell is the ratio of the output power of the device to the area of an adopted electrode, and is an important index for measuring the electricity generating efficiency of the microbial fuel cell. In the experiment, a 30-channel USB data acquisition card of Beijing Ribobowawa company is adopted to acquire the closed-circuit voltage of each MFC device, and the closed-circuit voltage of each battery device is recorded every 1 h. The calculation formula of the output power density is obtained by ohm's law and a related electrical formula:
wherein P closed represents the output power density, E represents the output voltage of the battery device, R external represents the external resistance of the battery device, and S electrode represents the area of the carbon sheet electrode. The external resistance is known to be 5k omega, and the electrode area is known to be 4cm2. The resulting power density curve is shown in FIG. 8, which illustrates Bacillus cereus (Bacillus cereu)s) DIF1 has better power generation capability and can be used for microbial fuel cells.
From the results, the bacillus cereus (Bacillus cereus) DIF1 is separated from the meishan iron ore, the strong Fe (III) and Cr (VI) ion reduction function of the bacillus cereus DIF1 is found, and the bacillus cereus DIF1 can also be used as an electricity-producing bacterium to be applied to a microbial battery, so that the application research thought of the bacillus cereus (Bacillus cereus) DIF1 on the function aspect is widened, a new material is provided for the iron reducing bacterium on the aspect of environmental pollutant treatment, and the application value is strong.
Sequence listing
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<120> bacillus cereus DIF1, microbial agent produced by same and application of microbial agent
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gcagtcgagc gaatggatta agaagcttgc tcttatgaag ttagcggcgg acgggtgagt 60
aacacgtggg taacctgccc ataagactgg gataactccg ggaaaccggg gctaataccg 120
gataacattt tgaaccgcat ggttcgaaat tgaaaggcgg cttcggctgt cacttatgga 180
tggacccgcg tcgcattagc tagttggtga ggtaacggct caccaaggca acgatgcgta 240
gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300
aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360
tgatgaaggc tttcgggtcg taaaactctg ttgttaggga agaacaagtg ctagttgaat 420
aagctggcac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg 480
cggtaatacg taggtggcaa gcgttatccg gaattattgg gcgtaaagcg cgcgcaggtg 540
gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg 600
agacttgagt gcagaagagg aaagtggaat tccatgtgta gcggtgaaat gcgtagagat 660
atggaggaac accagtggcg aaggcgactt tctggtctgt aactgacact gaggcgcgaa 720
agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct 780
aagtgttaga gggtttccgc cctttagtgc tgaagttaac gcattaagca ctccgcctgg 840
ggagtacggc cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 900
gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaaa 960
accctagaga tagggcttct ccttcgggag cagagtgaca ggtggtgcat ggttgtcgtc 1020
agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt 1080
gccatcatta agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg 1140
atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacggt 1200
acaaagagct gcaagaccgc gaggtggagc taatctcata aaaccgttct cagttcggat 1260
tgtaggctgc aactcgccta catgaagctg gaatcgctag taatcgcgga tcagcatgcc 1320
gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag gagtttgtaa 1380
cacccggaaa gtcggtgggg taaacctttt ggagccag 1418
Claims (6)
1. Bacillus cereus strainBacillus cereus) DIF1, which is preserved in China center for type culture Collection with the preservation time of 2018, 5 months and 14 days, and the preservation number is CCTCC NO: m2018274.
2. Use of bacillus cereus DIF1 according to claim 1 for dissimilatory reduction of fe (iii).
3. The use of bacillus cereus DIF1 as claimed in claim 1 for the reduction of hexavalent chromium.
4. A microbial agent produced using bacillus cereus DIF1 of claim 1, wherein the microbial agent is produced by fermentation of bacillus cereus DIF 1.
5. The method for culturing Bacillus cereus DIF1 according to claim 1, wherein the method comprises inoculating Bacillus cereus DIF1 in LB liquid medium and culturing with shaking.
6. Use of bacillus cereus DIF1 according to claim 1 or the microbial agent of claim 4 in a microbial fuel cell.
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| CN110951642B (en) * | 2019-12-18 | 2022-09-09 | 东南大学 | Method for reducing and adsorbing heavy metal chromium by fixed microorganisms |
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